Rust Resistance Gene

ABSTRACT

The present invention relates to new transporter polypeptides, and genes encoding therefor, which can be used to confer upon a plant resistance to one or more biotrophic fungal pathogens.

FIELD OF THE INVENTION

The present invention relates to new transporter polypeptides, and genesencoding therefor, which can be used to confer upon a plant resistanceto one or more biotrophic fungal pathogens.

BACKGROUND OF THE INVENTION

Numerous genes conferring resistance to pathogens have been identifiedand used in plant breeding. However, single-gene pathogen resistance inplants often becomes ineffective due to the emergence of new virulentraces of the disease agent. In contrast, durable disease resistance inplants is generally thought to be controlled by multiple genes. A fewrust resistance genes have been isolated and cloned from wheat (Feuilletet al., 2003; Huang et al., 2003; Cloutier et al., 2007) and othercereals (Collins et al., 1999; Brueggeman et al., 2002) and arepredominantly from the nucleotide binding site-leucine rich repeat(NB-LRR) class of major resistance (R) genes. For example, three wheat Rgenes (Lr1, Lr10 and Lr21) that provide protection against the wheatleaf rust fungus, Puccinia triticina, have been cloned (Somers et al.,2004; Hayden et al., 2008; Manly et al., 2001). One exception is thebarley Rpg1 rust resistance gene which encodes a protein kinase. Thesegenes encode gene-for-gene resistance against single pathogens andgenerally lead to strong, hypersensitive responses in the plant tissuesupon infection.

In contrast, rust resistance genes in wheat (Triticum aestivum L.) suchas Lr34, located on the chromosome arm 7DS, confer a broad spectrum anddurable adult plant resistance against several obligate biotrophicpathogens including fungi from the Ascomycetes and Basidiomycetes. Theseinclude leaf rust, stripe rust, stem rust and powdery mildew andtherefore the Lr34 gene has been widely deployed in wheat breedingdespite its weaker, non-hypersensitive response phenotype (Dyck, 1977and 1987; German and Kolmer, 1992; Bossolini et al., 2006; Spielmeyer etal., 2008). Cultivars with the resistance locus Lr34 such as Frontanahave had effective durable resistance to the leaf rust fungus Pucciniatriticina Eriks (Dyck et al., 1966; Singh and Rajaram, 1994). To date,isolates of P. triticina with complete virulence to Lr34 have not beendetected (Kolmer et al., 2003). The Lr34 gene was recently cloned andshown to encode a protein in the ABC transporter family (Krattinger etal., 2009), although its function as a transporter was not demonstrated.Lr34 resistance has remained genetically inseparable from the genedesignated Yr18 that confers resistance to stripe rust (P. striiformis)(Singh, 1992; McIntosh, 1992). Co-segregation of Lr34/Yr18 with othertraits such as leaf tip necrosis (Ltn1) in adulkt plant stage, powderymildew (recently designated Pm38), tolerance to barley yellow dwarfvirus (Bdv 1) and spot blotch (Bipolaris sorokiniana) have beendocumented (Singh, 1992a,b; McIntosh, 1992; Joshi et al., 2004;Spielmeyer et al., 2005; Liang et al., 2006), and these phenotypes areall thought to be conferred by the Lr34 resistance polypeptide.

A second gene that confers broad spectrum, adult plant resistanceagainst several obligate biotrophic pathogens is Lr67, located onchromosome 4DL in wheat, and found in a few wheat accessions such asRL6077 (Herrera-Foessel et al., 2011). In contrast to Lr34, the Lr67gene has not been widely used to produce resistant cultivars forcommercial wheat production. Although an initial report (Dyck et al.,(1994) based on plant phenotypes suggested that the resistance gene inRL6077 might be a translocated Lr34, this was subsequently shown not tobe the case (Herrera-Foessel et al., 2011). After mapping the gene intwo segregating populations, Hiebert et al., (2010) designated the genein RL6077 as Lr67. Although Lr67 also leads to leaf tip necrosis andprovides a partial, broad spectrum, adult plant resistance to leaf rustand stripe rust like Lr34, these are clearly different genes.

There is a need to determine the molecular basis of genes such as Lr67that provide quantitative non-race-specific, adult plant pathogenresistance-type or partial resistance to a broad spectrum of pathogens.

SUMMARY OF THE INVENTION

The present inventors have identified new transporter polypeptides, andgenes encoding therefor, which can be used to confer upon a plantresistance to one or more biotrophic fungal pathogens.

In a first aspect, the present invention provides a recombinant cellcomprising an exogenous polynucleotide encoding a polypeptide which ischaracterised by one or more or all of:

i) when expressed in a plant, the polypeptide confers upon the plantresistance to one or more biotrophic fungal pathogen(s), preferably toone or more or all of leaf rust, stripe rust, stem rust and powderymildew,

ii) when expressed in a cell, the polypeptide is not as active attransporting glucose across a membrane of the cell as a polypeptidewhich comprises amino acids having a sequence as provided in SEQ IDNO:4,

iii) when expressed in a cell, the polypeptide is active as a sugartransporter,

iv) the polypeptide comprises amino acids having a sequence as providedin SEQ ID NO:1 or an amino acid sequence which is at least 40% identicalto SEQ ID NO:1 or a biologically active fragment thereof, and

v) the polypeptide does not comprise a glycine at a positioncorresponding to amino acid number 144 of SEQ ID NO:1, preferably thepolypeptide comprises an amino acid other than glycine at the positioncorresponding to amino acid number 144 of SEQ ID NO:1,

wherein the polynucleotide is operably linked to a promoter capable ofdirecting expression of the polynucleotide in the cell.

In a preferred embodiment, the polypeptide at least has features i) andiv), i), ii) and iv), ii) and iv), or iv) and v), more preferablyfeatures i), iv) and v), i), ii), iv) and v), or i), iv) and v).

In an embodiment, the one or more biotrophic fungal pathogen(s) is arust or a mildew or both a rust and a mildew. Examples of biotrophicfungi include, but are not limited to, Blumeria graminis f. sp. tritici,Fusarium graminearum, Bipolaris sorokiniana, Erysiphe graminis f. sp.tritici, Puccinia graminis f. sp. tritici, Puccinia striiformis,Puccinia hordei and Puccinia recondita f. sp. tritici.

In an embodiment, the cell is a plant or yeast cell. More preferably,the cell is a plant cell. In an embodiment, the plant cell is a cerealplant cell such as a wheat plant cell. In another embodiment, the plantcell is a grape cell.

In an embodiment, the promoter directs gene expression in a leaf and/orstem cell.

Preferably, if the polypeptide does not comprise a glycine at a positioncorresponding to amino acid number 144 of SEQ ID NO:1, the polypeptidecomprises an amino acid sequence which is at least 40% identical to oneor more or all of SEQ ID NO's 1, 4 or 7 to 9, or a biologically activefragment of one or more thereof.

In a preferred embodiment, the polypeptide comprises an amino acid atthe position corresponding to amino acid number 144 of SEQ ID NO:1,wherein the amino acid is selected from the group consisting ofarginine, lysine and histidine.

In a further preferred embodiment, the polypeptide does not comprise avaline at a position corresponding to amino acid number 387 of SEQ IDNO:1, preferably the polypeptide comprises an amino acid other thanvaline at the position corresponding to amino acid number 387 of SEQ IDNO:1. More preferably, the polypeptide comprises an amino acid at theposition corresponding to amino acid number 387 of SEQ ID NO:1, whereinthe amino acid is selected from the group consisting of leucine,isoleucine, methionine, alanine and phenylalanine.

In an embodiment, the exogenous polynucleotide is integrated into thegenome of the cell.

In a further embodiment, the polypeptide comprises amino acids having asequence as provided in SEQ ID NO:1 or an amino acid sequence which isat least 80% identical, at least 90% identical, or at least 95%identical to SEQ ID NO:1, or a biologically active fragment thereof.

In an embodiment, the polypeptide comprises 12 transmembrane domains.

In another aspect, the present invention provides a transgenic plantcomprising cells of the invention, wherein the transgenic plant istransgenic for the exogenous polynucleotide.

In a preferred embodiment, each of the somatic cells of the plantcomprise the exogenous polynucleotide.

In a further preferred embodiment, the plant has enhanced resistance toone or more biotrophic fungal pathogen(s), preferably to a rust, amildew or both a rust and a mildew, more preferably to one or more orall of leaf rust, stripe rust, stem rust and powdery mildew, whencompared to an isogenic plant lacking the exogenous polynucleotide.

In a further embodiment, the plant has enhanced resistance to one ormore biotrophic fungal pathogen(s) at the seedling stage of growth.

In another embodiment, the plant comprises one or more further exogenouspolynucleotides encoding a plant pathogen resistance polypeptide otherthan an Lr67 polypeptide, preferably an Lr34 polypeptide, an Sr33polypeptide or an Sr35 polypeptide. Further plant pathogen resistancepolypeptides include, but are not limited to, Lr1, Lr3, Lr2a, Lr3ka,Lr11, Lr13, Lr16, Lr17, Lr18, Lr21, and LrB.

Preferably, the plant is a cereal plant. Examples of transgenic cerealplants of the invention include, but are not limited to wheat, barley,maize, rice, oats and triticale. In a particularly preferred embodiment,the plant is wheat. In another embodiment, the plant is a grapevine.

In an embodiment, the promotyer directs gene expression in an aerialpart of the plant such as the leaves and/or the stems.

Preferably, the plant is homozygous for the exogenous polynucleotide(s).

In a further embodiment, the plant is growing in a field.

Also provided is a population of at least 100 plants of the inventiongrowing in a field.

In another aspect, the present invention provides a process fordetermining whether a polypeptide confers resistance or susceptibilityto one or more biotrophic fungal pathogen(s), preferably a rust, amildew or both a rust and a mildew, more preferably to one or more orall of leaf rust, stripe rust, stem rust and powdery mildew, comprising:

i) obtaining a polynucleotide operably linked to a promoter, thepolynucleotide encoding the polypeptide, wherein the polypeptidecomprises amino acids having a sequence as provided in SEQ ID NO:1 or anamino acid sequence which is at least 40% identical to one SEQ ID NO:1,or a biologically active fragment thereof,

ii) introducing the polynucleotide into a plant,

iii) determining whether the level of resistance or susceptibility tothe one or more biotrophic fungal pathogen(s), preferably a rust, amildew or both a rust and a mildew, more preferably to one or more orall of leaf rust, stripe rust, stem rust and powdery mildew, isincreased or decreased relative to an isogenic plant lacking thepolynucleotide, and

iv) optionally, if the level of resistance or susceptibility isincreased, selecting a polynucleotide encoding the polypeptide whichwhen expressed confers resistance or susceptibility to the one or morebiotrophic fungal pathogen(s), preferably a rust, a mildew or both arust and a mildew, more preferably to one or more or all of leaf rust,stripe rust, stem rust and powdery mildew.

In an embodiment, one or more of the following apply to the process,

a) the polynucleotide comprises nucleotides having a sequence asprovided in SEQ ID NO:2 or SEQ ID NO:3, a sequence which is at least 40%identical to one or both of SEQ ID NO:2 and SEQ ID NO:3, or a sequencewhich hybridizes to one or both of SEQ ID NO:2 and SEQ ID NO:3,

b) the plant is a cereal plant such as a wheat plant or grapevine plant,

c) the polypeptide is a plant polypeptide or mutant thereof, and

d) step ii) further comprises stably integrating the polynucleotideoperably linked to a promoter into the genome of the plant

e) the polypeptide is characterised by one or more of the featuresdefined above in relation to a cell of the invention.

In another aspect, the present invention provides a substantiallypurified and/or recombinant polypeptide which is characterised by one ormore or all of:

i) when expressed in a plant, the polypeptide confers upon the plantresistance to one or more biotrophic fungal pathogen(s), preferably arust, a mildew or both a rust and a mildew, more preferably to one ormore or all of leaf rust, stripe rust, stem rust and powdery mildew,

ii) when expressed in a cell, the polypeptide is not as active attransporting glucose across a membrane of the cell as a polypeptidewhich comprises amino acids having a sequence as provided in SEQ IDNO:4,

iii) when expressed in a cell, the polypeptide is active as a sugartransporter,

iv) the polypeptide comprises amino acids having a sequence as providedin SEQ ID NO:1 or an amino acid sequence which is at least 40% identicalto SEQ ID NO:1, or a biologically active fragment thereof, and

v) the polypeptide does not comprise a glycine at a positioncorresponding to amino acid number 144 of SEQ ID NO:1, preferably thepolypeptide comprises an amino acid other than glycine at the positioncorresponding to amino acid number 144 of SEQ ID NO:1.

In a preferred embodiment, the polypeptide is characterised by one ormore of the features defined above in relation to a cell of theinvention.

In another aspect, the polypeptide comprises amino acids having asequence as provided in SEQ ID NO:1 or an amino acid sequence which isat least 80% identical, at least 90% identical, or at least 95%identical, to SEQ ID NO:1, or a biologically active fragment thereof.

In an embodiment, a polypeptide of the invention is a fusion proteinfurther comprising at least one other polypeptide sequence. The at leastone other polypeptide may be, for example, a polypeptide that enhancesthe stability of a polypeptide of the present invention, or apolypeptide that assists in the purification or detection of the fusionprotein.

In a further aspect, the present invention provides an isolated and/orexogenous polynucleotide comprising nucleotides having a sequence asprovided in SEQ ID NO:2 or SEQ ID NO:3, a sequence which is at least 40%identical to one or both of SEQ ID NO:2 and SEQ ID NO:3, a sequenceencoding a polypeptide of the invention, or a sequence which hybridizesto one or both of SEQ ID NO:2 and SEQ ID NO:3.

In another aspect, the present invention provides a chimeric vectorcomprising the polynucleotide of the invention.

Preferably, the polynucleotide is operably linked to a promoter.

In a further aspect, the present invention provides a recombinant cellcomprising an exogenous polynucleotide of the invention and/or a vectorof the invention.

The cell can be any cell type such as, but not limited to, a plant cell,a bacterial cell, an animal cell or a yeast cell.

Preferably, the cell is a plant cell. More preferably, the plant cell isa cereal plant cell. Even more preferably, the cereal plant cell is awheat cell. In another embodiment, the plant cell is a grape cell.

In a further aspect, the present invention provides a method ofproducing the polypeptide of the invention, the method comprisingexpressing in a cell or cell free expression system the polynucleotideof the invention.

Preferably, the method further comprises isolating the polypeptide.

In another aspect, the present invention provides a method of producingthe cell of the invention, the method comprising the step of introducingthe polynucleotide of the invention, or a vector of the invention, intoa cell.

Preferably, the cell is a plant cell.

In another aspect, the present invention provides a method of producinga transgenic plant of the invention, the method comprising the steps of

i) introducing a polynucleotide of the invention and/or a vector of theinvention into a cell of a plant,

ii) regenerating a transgenic plant from the cell, and

iii) optionally harvesting seed from the plant, and/or

iv) optionally producing one or more progeny plants from the transgenicplant, thereby producing the transgenic plant.

In another aspect, the present invention provides a method of producinga plant which has integrated into its genome a polynucleotide encoding apolypeptide of the invention, the method comprising the steps of

i) crossing two parental plants, wherein at least one plant comprises apolynucleotide encoding the polypeptide,

ii) screening one or more progeny plants from the cross for the presenceor absence of the polynucleotide, and

iii) selecting a progeny plant which comprise the polynucleotide,thereby producing the plant.

In an embodiment, the polypeptide comprises amino acids having asequence as provided in SEQ ID NO:1 or an amino acid sequence which isat least 40% identical to SEQ ID NO:1, or a biologically active fragmentthereof, and wherein when expressed in the plant, the polypeptideconfers upon the plant resistance to one or more biotrophic fungalpathogen(s), preferably a rust, a mildew or both a rust and a mildew,preferably to one or more or all of leaf rust, stripe rust, stem rustand powdery mildew.

In an embodiment, at least one of the parental plants is a tetraploid orhexaploid wheat plant. In another embodiment, the parental plant is agrapevine.

In another embodiment, step ii) comprises analysing a sample comprisingDNA from the plant for the polynucleotide.

In a further embodiment, step iii) comprises

i) selecting progeny plants which are homozygous for the polynucleotide,and/or

ii) analysing the plant or one or more progeny plants thereof forresistance to one or more biotrophic fungal pathogen(s), preferably arust, a mildew or both of a rust and a mildew, more preferably to one ormore or all of leaf rust, stripe rust, stem rust and powdery mildew.

In another embodiment, the method further comprises

iv) backcrossing the progeny of the cross of step i) with plants of thesame genotype as a first parent plant which lacked a polynucleotideencoding the polypeptide for a sufficient number of times to produce aplant with a majority of the genotype of the first parent but comprisingthe polynucleotide, and

iv) selecting a progeny plant which has resistance to one or morebiotrophic fungal pathogen(s), preferably a rust, a mildew or both arust and a mildew, more preferably to one or more or all of leaf rust,stripe rust, stem rust and powdery mildew.

In an embodiment, the method further comprises the step of analysing theplant for at least one other genetic marker.

Also provided is a plant produced using the method of the invention.

In another aspect, the present invention provides for the use of thepolynucleotide of the invention, or a vector of the invention, toproduce a recombinant cell and/or a transgenic plant.

In an embodiment, the transgenic plant has enhanced resistance to one ormore biotrophic fungal pathogen(s), preferably a rust, a mildew or botha rust and a mildew, more preferably to one or more or all of leaf rust,stripe rust, stem rust and powdery mildew, when compared to an isogenicplant lacking the exogenous polynucleotide and/or vector.

In a further aspect, the present invention provides a method foridentifying a plant comprising a polynucleotide encoding a polypeptideof the invention, the method comprising the steps of

i) obtaining a nucleic acid sample from a plant, and

ii) screening the sample for the presence or absence of thepolynucleotide.

In an embodiment, the presence of the polynucleotide indicates that theplant has enhanced resistance to one or more biotrophic fungalpathogen(s), preferably a rust, a mildew or both a rust and a mildew,more preferably to one or more or all of leaf rust, stripe rust, stemrust and powdery mildew, when compared to an isogenic plant lacking theexogenous polynucleotide.

In an embodiment, a genomic region encompassing the polynucleotide isamplified and the amplification product sequenced to determine if itencodes the polypeptide. Primers useful for the amplification, anduseful for the sequencing, can readily be designed by the skilledperson.

In a further embodiment, the method identifies a transgenic plant of theinvention.

In embodiment, the method further comprises producing a plant from aseed before step i).

Also provided is a plant part of the plant of the invention.

In an embodiment, the plant part is a seed that comprises an exogenouspolynucleotide which encodes a polypeptide of the invention.

In a further aspect, the present invention provides a method ofproducing a plant part, the method comprising,

a) growing a plant of the invention, and

b) harvesting the plant part.

In another aspect, the present invention provides a method of producingflour, wholemeal, starch or other product obtained from seed, the methodcomprising;

a) obtaining seed of the invention, and

b) extracting the flour, wholemeal, starch or other product.

In a further aspect, the present invention provides a product producedfrom a plant of the invention and/or a plant part of the invention.

In an embodiment, the part is a seed.

In an embodiment, the product is a food product or beverage product.Examples include, but are not limited to;

i) the food product being selected from the group consisting of: flour,starch, leavened or unleavened breads, pasta, noodles, animal fodder,breakfast cereals, snack foods, cakes, malt, beer, pastries and foodscontaining flour-based sauces, or

ii) the beverage product being beer or malt.

In an alternative embodiment, the product is a non-food product.Examples include, but are not limited to, films, coatings, adhesives,building materials and packaging materials.

In a further aspect, the present invention provides a method ofpreparing a food product of the invention, the method comprising mixingseed, or flour, wholemeal or starch from the seed, with another foodingredient.

In another aspect, the present invention provides a method of preparingmalt, comprising the step of germinating seed of the invention.

Also provided is the use of a plant of the invention, or part thereof,as animal feed, or to produce feed for animal consumption or food forhuman consumption.

In a further aspect, the present invention provides a compositioncomprising one or more of a polypeptide of the invention, apolynucleotide of the invention, a vector of the invention, or arecombinant cell of the invention, and one or more acceptable carriers.

In a further aspect, the present invention provides a method ofidentifying a compound that binds to a polypeptide comprising aminoacids having a sequence as provided in SEQ ID NO:1 or an amino acidsequence which is at least 40% identical to SEQ ID NO:1, a biologicallyactive fragment thereof, the method comprising:

i) contacting the polypeptide with a candidate compound, and

ii) determining whether the compound binds the polypeptide.

In an embodiment, the polypeptide is embedded in a cell membrance,preferably the membrane of a plant cell.

In a further aspect, the present invention provides a method ofidentifying a compound which is transported across a cell membrane by apolypeptide comprising amino acids having a sequence as provided in SEQID NO:1 or SEQ ID NO:4 or an amino acid sequence which is at least 40%identical to one or both of SEQ ID NO:1 or SEQ ID NO:4, or abiologically active fragment thereof, the method comprising:

i) contacting the polypeptide embedded in a cell membrance, preferablythe membrane of a plant cell, with a candidate compound,

ii) determining whether the compound is transported from one side of themembrane to the other by the polypeptide.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1—Comparative genomics and mutant analysis.

FIG. 2—Deleted Hsp70 gene is completely linked to Lr67.

FIG. 3—Comparative genomics and mutant analysis including SUT and PIP.

FIG. 4—Nucleotide changes found in Lr67 variants.

FIG. 5—Amino acid sequence (514 amino acids, SEQ ID NO:1) of the SUTpolypeptide encoded by the Lr67 (resistant) allele of wheat. Thearginine at position 144 in the predicted fourth trans-membrane domainand the leucine at position 387 distinguish the resistant Lr67 from thesusceptible Lr67 polypeptide.

FIG. 6—Nucleotide sequence (SEQ ID NO:3) of the cDNA corresponding tothe Lr67 resistant allele. Two SNP positions that distinguish +/−Lr67are at positions 514 and 1243; these result in amino acid substitutionsin the encoded polypeptides. The translation start codon is at position85-87 and the translation stop codon is at position 1627-1629.

FIG. 7—Amino acid sequence (SEQ ID NO:4) of the Lr67 susceptiblepolypeptide (SUT).

FIG. 8—Nucleotide sequence (SEQ ID NO:6) of the cDNA corresponding tothe Lr67 susceptible allele SUT.

FIG. 9—Alignment of the amino acid sequences of the wheat Lr67(resistant) polypeptide (SEQ ID NO:1) with the homologous Arabidopsisthaliana polypeptide (Arath; from Genbank Accession No. NP_198006, 526amino acids) (SEQ ID NO:7). Stars indicate an identical amino acidresidue in that position, while “+” indicates a similar amino acid atthat position.

FIG. 10—Alignment of the amino acid sequences of the wheat Lr67(resistant) polypeptide (SEQ ID NO:1) with the homologous rice (Oryzasativa) polypeptide (Genbank Accession No. AAQ24871, 515 amino acids)(SEQ ID NO:8). Stars indicate an identical amino acid residue in thatposition, a “+” indicates a similar amino acid at that position.

FIG. 11—Glucose uptake of yeast cells expressing Lr67 (resistant) andLr67 (susceptible) proteins.

FIG. 12—Kinetics of glucose uptake in yeast expressing Lr67(susceptible).

FIG. 13—Effect of variant aminio acids on glucose transport by Lr67proteins.

FIG. 14—Nucleotide sequence of a genomic fragment corresponding to theprotein coding region of a Lr67 (susceptible) gene. The sequence startswith the translation start codon ATG and ends with the translation stopTGA. The two introns in the protein coding region are nucleotides137-876 (Intron 1, 740nt) and 1197-3154 (Intron 2, 1958nt).

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—Wheat Lr67 (resistant) protein.

SEQ ID NO: 2—Open reading frame encoding wheat Lr67 (resistant) protein.

SEQ ID NO: 3—cDNA encoding wheat Lr67 (resistant) protein.

SEQ ID NO:4—Wheat Lr67 (susceptible) protein.

SEQ ID NO: 5—Open reading frame encoding wheat Lr67 (susceptible)protein.

SEQ ID NO: 6—cDNA encoding wheat Lr67 (susceptible) protein.

SEQ ID NO:7—Arabidopsis thaliana Lr67 protein.

SEQ ID NO:8—Rice (Oryza sativa)Lr67 protein.

SEQ ID NO:9—Grapevine (Vitis vinifera) Lr67 protein.

SEQ ID NO:10—Gene encoding wheat Lr67 (susceptible) protein.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, plant molecular biology, protein chemistry, andbiochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Polypeptides

The present invention relates to polypeptides which, when expressed in aplant, confer upon the plant resistance to one or more biotrophic fungalpathogen(s), preferably to one or more or all of leaf rust, stripe rust,stem rust and powdery mildew. The present invention also relates topolypeptides which, when expressed in a cell, are not as active attransporting glucose across a membrane of the cell as a polypeptidewhich comprises amino acids having a sequence as provided in SEQ IDNO:4. In addition, the present invention also relates to polypeptideswhich, when expressed in a cell, act as a sugar transporter. In apreferred embodiment, the polypeptide is encoded by an allele or variantof an Lr67 gene which confers plant resistance to one or more biotrophicfungal pathogen(s). Examples of such polypeptides include, but are notlimited to, those comprising an amino acid sequence as provided in SEQID NO:1. The polypeptide of the invention confers enhanced resistance toone or more biotrophic fungal pathogen(s) when compared to an isogenicplant lacking a polynucleotide encoding the polypeptide.

As used herein, the term “Lr67” relates to a protein family which sharehigh primary amino acid sequence identity, for example at least 40%,least 80%, at least 90%, or at least 95% identity with one or more ofthe amino acid sequences provided as SEQ ID NO:1, 4 or 7 to 9,preferably SEQ ID NO:1. The present inventors have determined that somevariants of the Lr67 protein family, when expressed in a plant, conferupon the plant resistance to one or more biotrophic fungal pathogen(s),preferably to one or more or all of leaf rust, stripe rust, stem rustand powdery mildew. An example of such a variant comprises an amino acidsequence provided as SEQ ID NO:1. Thus, variants which confer resistanceare referred to herein as Lr67 (resistant) polypeptides, whereas thosewhich do not (such as comprising the amino acid sequence provided as SEQID NO:4) are referred to herein as Lr67 (susceptible) polypeptides. In apreferred embodiment, Lr67 (resistant) proteins do not comprise aglycine at a position corresponding to amino acid number 144 of SEQ IDNO:1, preferably the polypeptide comprises an amino acid other thanglycine at the position corresponding to amino acid number 144 of SEQ IDNO:1. The amino acid at position 144 is preferably a charged amino acidsuch as arginine, lysine or histidine.

As used herein, “resistance” is a relative term in that the presence ofa polypeptide of the invention (i) reduces the disease symptoms of aplant comprising the gene (R (resistant) gene) that confers resistance,relative to a plant lacking the R gene, and/or (ii) reduces pathogenreproduction or spread on a plant or within a population of plantscomprising the R gene. Resistance as used herein is relative to the“susceptible” response of a plant to the same pathogen. Typically, thepresence of the R gene improves at least one production trait of a plantcomprising the R gene when infected with the pathogen, such as grainyield, when compared to an isogenic plant infected with the pathogen butlacking the R gene. The isogenic plant may have some level of resistanceto the pathogen, or may be classified as susceptible. Thus, the terms“resistance” and “enhanced resistance” are generally used hereininterchangeably. Furthermore, a polypeptide of the invention does notnecessarily confer complete pathogen resistance, for example when somesymptoms still occur or there is some pathogen reproduction on infectionbut at a reduced amount within a plant or a population of plants.Resistance may occur at only some stages of growth of the plant, forexample in adult plants (fully grown in size) and less so, or not atall, in seedlings, or at all stages of plant growth. By using atransgenic strategy to express an Lr67 polypeptide in a plant, the plantof the invention can be provided with resistance throughout its growthand development. Enhanced resistance can be determined by a number ofmethods known in the art such as analysing the plants for the amount ofpathogen and/or analysing plant growth or the amount of damage ordisease symptoms to a plant in the presence of the pathogen, andcomparing one or more of these parameters to an isogenic plant lackingan exogenous gene encoding a polypeptide of the invention.

As used herein, a “sugar transporter” is a membrane bound protein whichfacilitiates the movement of a sugar across a membrane, for example fromoutside of a cell into the cell, or in the opposite direction frominside a cell to outside, or across a membrane of a subcellularorganelle within the cell. The facilitation may be active, using anenergy source such as from an ionic gradient across the membrane, orpassive. For Lr67 (susceptible) proteins the sugar can be glucose. In anembodiment, the sugar is a monosaccaharide, preferably a hexosemonosaccaharide or a pentose polysaccharide. In an embodiment, the sugarmay be modified such as a sugar alcohol or phosphorylated sugar.

As used herein, the phrase “not as active at transporting glucose acrossa membrane of the cell as a polypeptide which comprises amino acidshaving a sequence as provided in SEQ ID NO:4” means that the polypeptideof the invention has less than 50%, or less than 25%, or less than 10%,of the ability of a polypeptide which comprises amino acids having asequence as provided in SEQ ID NO:4 to transport glucose into a cellsuch as a yeast or plant cell. This can readily be determined asdescribed herein (see, for instance, FIG. 11 and associated experimentaldetails).

By “substantially purified polypeptide” or “purified polypeptide” wemean a polypeptide that has generally been separated from the lipids,nucleic acids, other peptides, and other contaminating molecules withwhich it is associated in its native state. Preferably, thesubstantially purified polypeptide is at least 90% free from othercomponents with which it is naturally associated. In an embodiment, thepolypeptide of the invention has an amino acid sequence which isdifferent to a naturally occurring Lr67 polypeptide i.e. is an aminoacid sequence variant.

Transgenic plants and host cells of the invention may comprise anexogenous polynucleotide encoding a polypeptide of the invention. Inthese instances, the plants and cells produce a recombinant polypeptide.The term “recombinant” in the context of a polypeptide refers to thepolypeptide encoded by an exogenous polynucleotide when produced by acell, which polynucleotide has been introduced into the cell or aprogenitor cell by recombinant DNA or RNA techniques such as, forexample, transformation. Typically, the cell comprises a non-endogenousgene that causes an altered amount of the polypeptide to be produced. Inan embodiment, a “recombinant polypeptide” is a polypeptide made by theexpression of an exogenous (recombinant) polynucleotide in a plant cell.

The terms “polypeptide” and “protein” are generally usedinterchangeably.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 400 aminoacids in length, and the GAP analysis aligns the two sequences over aregion of at least 400 amino acids. More preferably, the query sequenceis at least 500 amino acids in length and the GAP analysis aligns thetwo sequences over a region of at least 500 amino acids. Even morepreferably, the GAP analysis aligns two sequences over their entirelength, which for an Lr67 polypeptide is about 514 amino acid residues.

As used herein, a “biologically active fragment” is a portion of apolypeptide of the invention which maintains a defined activity of thefull-length polypeptide such as one or both of i) when expressed in aplant, such as wheat, confers (enhanced) resistance to one or morebiotrophic fungal pathogen(s), preferably to one or more or all of leafrust, stripe rust, stem rust and powdery mildew, and ii) when expressedin a cell, the polypeptide is active as a sugar transporter, preferablynot as active at transporting glucose across a membrane of the cell as apolypeptide which comprises amino acids having a sequence as provided inSEQ ID NO:4. Biologically active fragments can be any size as long asthey maintain the defined activity but are preferably at least 400 or atleast 500 amino acid residues long. Preferably, the biologically activefragment maintains at least 50%, at least 75% or at least 90%, of theactivity of the full length protein. In an embodiment, the biologicallyactive fragment comprises 12 transmembrane domains.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

In an embodiment, a polypeptide of the invention is not a naturallyoccurring polypeptide such consisting of an amino acid sequence providedas SEQ ID NO:1 or SEQ ID NO:4.

As used herein, the phrase “at a position corresponding to amino acidnumber” or variations thereof refers to the relative position of theamino acid compared to surrounding amino acids. In this regard, in someembodiments a polypeptide of the invention may have deletional orsubstitutional mutation which alters the relative positioning of theamino acid when aligned against, for instance, SEQ ID NO:1. For example,as shown in FIG. 9 amino acid number 178 of wheat Lr67 (resistant)corresponds to amino acid number 179 of the homologous Lr67 protein fromArabidopsis.

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid of the present invention, or by in vitro synthesis of thedesired polypeptide. Such mutants include, for example, deletions,insertions or substitutions of residues within the amino acid sequence.A combination of deletion, insertion and substitution can be made toarrive at the final construct, provided that the final peptide productpossesses the desired characteristics. Preferred amino acid sequencemutants have only one, two, three, four or less than 10 amino acidchanges relative to the reference wildtype polypeptide.

Mutant (altered) polypeptides can be prepared using any technique knownin the art, for example, using directed evolution or rational designstrategies (see below). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey have one or more of the following features i) when expressed in aplant, such as wheat, confer (enhanced) resistance to one or morebiotrophic fungal pathogen(s), preferably to one or more or all of leafrust, stripe rust, stem rust and powdery mildew, ii) when expressed in acell, the encoded polypeptide is not as active at transporting glucoseacross a membrane of the cell as a polypeptide which comprises aminoacids having a sequence as provided in SEQ ID NO:4, and iii) whenexpressed in a cell, the polypeptide is active as a sugar transporter.For instance with regard to i), the method may comprise producing atransgenic plant expressing the mutated/altered DNA and determining theeffect of the pathogen on the growth of the plant.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. Where it is derisable to maintain a certain activity it ispreferable to make no, or only conservative substitutions, at amino acidpositions which are highly conserved in the relevant protein family.Examples of conservative substitutions are shown in Table 1 under theheading of “exemplary substitutions”.

In a preferred embodiment a mutant/variant polypeptide has one or two orthree or four conservative amino acid changes when compared to anaturally occurring polypeptide. Details of conservative amino acidchanges are provided in Table 1. In a preferred embodiment, the changesare not in one or more of the motifs which are highly conserved betweenthe different polypeptides provided herewith, and/or not in the 12transmembrane helices of the Lr67 polypeptides. As the skilled personwould be aware, such minor changes can reasonably be predicted not toalter the activity of the polypeptide when expressed in a recombinantcell.

The primary amino acid sequence of a polypeptide of the invention can beused to design variants/mutants thereof based on comparisons withclosely related sugar transporter polypeptides (for example, as shown inFIGS. 9 and 10). As the skilled addressee will appreciate, residueshighly conserved amongst closely related proteins are less likely to beable to be altered, especially with non-conservative substitutions, andactivity maintained than less conserved residues (see above).

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. The polypeptides may bepost-translationally modified in a cell, for example by phosphorylation,which may modulate its activity. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

TABLE 1 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

Directed Evolution

In directed evolution, random mutagenesis is applied to a protein, and aselection regime is used to pick out variants that have the desiredqualities, for example, increased activity. Further rounds of mutationand selection are then applied. A typical directed evolution strategyinvolves three steps:

1) Diversification: The gene encoding the protein of interest is mutatedand/or recombined at random to create a large library of gene variants.Variant gene libraries can be constructed through error prone PCR (see,for example, Leung, 1989; Cadwell and Joyce, 1992), from pools of DNaseldigested fragments prepared from parental templates (Stemmer, 1994a;Stemmer, 1994b; Crameri et al., 1998; Coco et al., 2001) from degenerateoligonucleotides (Ness et al., 2002, Coco, 2002) or from mixtures ofboth, or even from undigested parental templates (Zhao et al., 1998;Eggert et al., 2005; Jézéquek et al., 2008) and are usually assembledthrough PCR. Libraries can also be made from parental sequencesrecombined in vivo or in vitro by either homologous or non-homologousrecombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber etal., 2001). Variant gene libraries can also be constructed bysub-cloning a gene of interest into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. Variant gene libraries can also be constructed bysubjecting the gene of interest to DNA shuffling (i.e., in vitrohomologous recombination of pools of selected mutant genes by randomfragmentation and reassembly) as broadly described by Harayama (1998).

2) Selection: The library is tested for the presence of mutants(variants) possessing the desired property using a screen or selection.Screens enable the identification and isolation of high-performingmutants by hand, while selections automatically eliminate allnonfunctional mutants. A screen may involve screening for the presenceof known conserved amino acid motifs. Alternatively, or in addition, ascreen may involve expressing the mutated polynucleotide in a hostorgansim or part thereof and assaying the level of activity.

3) Amplification: The variants identified in the selection or screen arereplicated many fold, enabling researchers to sequence their DNA inorder to understand what mutations have occurred.

Together, these three steps are termed a “round” of directed evolution.Most experiments will entail more than one round. In these experiments,the “winners” of the previous round are diversified in the next round tocreate a new library. At the end of the experiment, all evolved proteinor polynucleotide mutants are characterized using biochemical methods.

Rational Design

A protein can be designed rationally, on the basis of known informationabout protein structure and folding. This can be accomplished by designfrom scratch (de novo design) or by redesign based on native scaffolds(see, for example, Hellinga, 1997; and Lu and Berry, Protein StructureDesign and Engineering, Handbook of Proteins 2, 1153-1157 (2007)).Protein design typically involves identifying sequences that fold into agiven or target structure and can be accomplished using computer models.Computational protein design algorithms search the sequence-conformationspace for sequences that are low in energy when folded to the targetstructure. Computational protein design algorithms use models of proteinenergetics to evaluate how mutations would affect a protein's structureand function. These energy functions typically include a combination ofmolecular mechanics, statistical (i.e. knowledge-based), and otherempirical terms. Suitable available software includes IPRO (InterativeProtein Redesign and Optimization), EGAD (A Genetic Algorithm forProtein Design), Rosetta Design, Sharpen, and Abalone.

Polynucleotides and Genes

The present invention refers to various polynucleotides. As used herein,a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means apolymer of nucleotides, which may be DNA or RNA or a combinationthereof, and includes genomic DNA, mRNA, cRNA, and cDNA. Less preferredpolynucleotides include tRNA, siRNA, shRNA and hpRNA. It may be DNA orRNA of cellular, genomic or synthetic origin, for example made on anautomated synthesizer, and may be combined with carbohydrate, lipids,protein or other materials, labelled with fluorescent or other groups,or attached to a solid support to perform a particular activity definedherein, or comprise one or more modified nucleotides not found innature, well known to those skilled in the art. The polymer may besingle-stranded, essentially double-stranded or partly double-stranded.Basepairing as used herein refers to standard basepairing betweennucleotides, including G:U basepairs. “Complementary” means twopolynucleotides are capable of basepairing (hybridizing) along part oftheir lengths, or along the full length of one or both. A “hybridizedpolynucleotide” means the polynucleotide is actually basepaired to itscomplement. The term “polynucleotide” is used interchangeably hereinwith the term “nucleic acid”. Preferred polynucleotides of the inventionencode a polypeptide of the invention.

By “isolated polynucleotide” we mean a polynucleotide which hasgenerally been separated from the polynucleotide sequences with which itis associated or linked in its native state, if the polynucleotide isfound in nature. Preferably, the isolated polynucleotide is at least 90%free from other components with which it is naturally associated, if itis found in nature. Preferably the polynucleotide is not naturallyoccurring, for example by covalently joining two shorter polynucleotidesequences in a manner not found in nature (chimeric polynucleotide).

The present invention involves modification of gene activity and theconstruction and use of chimeric genes. As used herein, the term “gene”includes any deoxyribonucleotide sequence which includes a proteincoding region or which is transcribed in a cell but not translated, aswell as associated non-coding and regulatory regions. Such associatedregions are typically located adjacent to the coding region or thetranscribed region on both the 5′ and 3′ ends for a distance of about 2kb on either side. In this regard, the gene may include control signalssuch as promoters, enhancers, termination and/or polyadenylation signalsthat are naturally associated with a given gene, or heterologous controlsignals in which case the gene is referred to as a “chimeric gene”. Thesequences which are located 5′ of the coding region and which arepresent on the mRNA are referred to as 5′ non-translated sequences. Thesequences which are located 3′ or downstream of the coding region andwhich are present on the mRNA are referred to as 3′ non-translatedsequences. The term “gene” encompasses both cDNA and genomic forms of agene.

A “Lr67 gene” as used herein refers to a nucleotide sequence which ishomologous to an isolated Lr67 cDNA (such as provided in SEQ ID NO:3 andSEQ ID NO:6). As described herein, some alleles and variants of the Lr67gene family encode a protein that confers resistance to one or morebiotrophic fungal pathogen(s), preferably to one or more or all of leafrust, stripe rust, stem rust and powdery mildew (see, for example, SEQID NO:3). Lr67 genes include the naturally occurring alleles or variantsexisting in cereals such as wheat, as well as articifially producedvariants.

A genomic form or clone of a gene containing the transcribed region maybe interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences”, which may be eitherhomologous or heterologous with respect to the “exons” of the gene. An“intron” as used herein is a segment of a gene which is transcribed aspart of a primary RNA transcript but is not present in the mature mRNAmolecule. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA). Introns may contain regulatory elements such as enhancers. Asdescribed herein, the wheat Lr67 genes (both resistant and susceptiblealleles) contain two introns in their protein coding regions. “Exons” asused herein refer to the DNA regions corresponding to the RNA sequenceswhich are present in the mature mRNA or the mature RNA molecule in caseswhere the RNA molecule is not translated. An mRNA functions duringtranslation to specify the sequence or order of amino acids in a nascentpolypeptide. The term “gene” includes a synthetic or fusion moleculeencoding all or part of the proteins of the invention described hereinand a complementary nucleotide sequence to any one of the above. A genemay be introduced into an appropriate vector for extrachromosomalmaintenance in a cell or, preferably, for integration into the hostgenome.

As used herein, a “chimeric gene” refers to any gene that comprisescovalently joined sequences that are not found joined in nature.Typically, a chimeric gene comprises regulatory and transcribed orprotein coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. In anembodiment, the protein coding region of an Lr67 gene is operably linkedto a promoter or polyadenylation/terminator region which is heterologousto the Lr67 gene, thereby forming a chimeric gene. The term “endogenous”is used herein to refer to a substance that is normally present orproduced in an unmodified plant at the same developmental stage as theplant under investigation. An “endogenous gene” refers to a native genein its natural location in the genome of an organism. As used herein,“recombinant nucleic acid molecule”, “recombinant polynucleotide” orvariations thereof refer to a nucleic acid molecule which has beenconstructed or modified by recombinant DNA technology. The terms“foreign polynucleotide” or “exogenous polynucleotide” or “heterologouspolynucleotide” and the like refer to any nucleic acid which isintroduced into the genome of a cell by experimental manipulations.

Foreign or exogenous genes may be genes that are inserted into anon-native organism, native genes introduced into a new location withinthe native host, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. The term“genetically modified” includes introducing genes into cells bytransformation or transduction, mutating genes in cells and altering ormodulating the regulation of a gene in a cell or organisms to whichthese acts have been done or their progeny.

Furthermore, the term “exogenous” in the context of a polynucleotide(nucleic acid) refers to the polynucleotide when present in a cell thatdoes not naturally comprise the polynucleotide. The cell may be a cellwhich comprises a non-endogenous polynucleotide resulting in an alteredamount of production of the encoded polypeptide, for example anexogenous polynucleotide which increases the expression of an endogenouspolypeptide, or a cell which in its native state does not produce thepolypeptide. Increased production of a polypeptide of the invention isalso referred to herein as “over-expression”. An exogenouspolynucleotide of the invention includes polynucleotides which have notbeen separated from other components of the transgenic (recombinant)cell, or cell-free expression system, in which it is present, andpolynucleotides produced in such cells or cell-free systems which aresubsequently purified away from at least some other components. Theexogenous polynucleotide (nucleic acid) can be a contiguous stretch ofnucleotides existing in nature, or comprise two or more contiguousstretches of nucleotides from different sources (naturally occurringand/or synthetic) joined to form a single polynucleotide. Typically suchchimeric polynucleotides comprise at least an open reading frameencoding a polypeptide of the invention operably linked to a promotersuitable of driving transcription of the open reading frame in a cell ofinterest.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 1,200nucleotides in length, and the GAP analysis aligns the two sequencesover a region of at least 1,200 nucleotides. Even more preferably, thequery sequence is at least 1,500 nucleotides in length and the GAPanalysis aligns the two sequences over a region of at least 1,500nucleotides. Even more preferably, the GAP analysis aligns two sequencesover their entire length.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

In a further embodiment, the present invention relates topolynucleotides which are substantially identical to those specificallydescribed herein. As used herein, with reference to a polynucleotide theterm “substantially identical” means the substitution of one or a few(for example 2, 3, or 4) nucleotides whilst maintaining at least oneactivity of the native protein encoded by the polynucleotide. Inaddition, this term includes the addition or deletion of nucleotideswhich results in the increase or decrease in size of the encoded nativeprotein by one or a few (for example 2, 3, or 4) amino acids whilstmaintaining at least one activity of the native protein encoded by thepolynucleotide.

The present invention also relates to the use of oligonucleotides, forinstance in methods of screening for a polynucleotide of, or encoding apolypeptide of, the invention. As used herein, “oligonucleotides” arepolynucleotides up to 50 nucleotides in length. The minimum size of sucholigonucleotides is the size required for the formation of a stablehybrid between an oligonucleotide and a complementary sequence on anucleic acid molecule of the present invention. They can be RNA, DNA, orcombinations or derivatives of either. Oligonucleotides are typicallyrelatively short single stranded molecules of 10 to 30 nucleotides,commonly 15-25 nucleotides in length. When used as a probe or as aprimer in an amplification reaction, the minimum size of such anoligonucleotide is the size required for the formation of a stablehybrid between the oligonucleotide and a complementary sequence on atarget nucleic acid molecule. Preferably, the oligonucleotides are atleast 15 nucleotides, more preferably at least 18 nucleotides, morepreferably at least 19 nucleotides, more preferably at least 20nucleotides, even more preferably at least 25 nucleotides in length.Oligonucleotides of the present invention used as a probe are typicallyconjugated with a label such as a radioisotope, an enzyme, biotin, afluorescent molecule or a chemiluminescent molecule.

The present invention includes oligonucleotides that can be used as, forexample, probes to identify nucleic acid molecules, or primers toproduce nucleic acid molecules. Probes and/or primers can be used toclone homologues of the polynucleotides of the invention from otherspecies. Furthermore, hybridization techniques known in the art can alsobe used to screen genomic or cDNA libraries for such homologues.

Polynucleotides and oligonucleotides of the present invention includethose which hybridize under stringent conditions to one or more of thesequences provided as SEQ ID NO's: 2, 3, 5 or 6. As used herein,stringent conditions are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% NaDodSO₄ at 50° C.; (2) employ during hybridisation adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone,50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1%SDS.

Polynucleotides of the present invention may possess, when compared tonaturally occurring molecules, one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis on the nucleic acid). A variant of a polynucleotide or anoligonucleotide of the invention includes molecules of varying sizes of,and/or are capable of hybridising to, the wheat genome close to that ofthe reference polynucleotide or oligonucleotide molecules definedherein. For example, variants may comprise additional nucleotides (suchas 1, 2, 3, 4, or more), or less nucleotides as long as they stillhybridise to the target region. Furthermore, a few nucleotides may besubstituted without influencing the ability of the oligonucleotide tohybridise to the target region. In addition, variants may readily bedesigned which hybridise close to, for example to within 50 nucleotides,the region of the plant genome where the specific oligonucleotidesdefined herein hybridise. In particular, this includes polynucleotideswhich encode the same polypeptide or amino acid sequence but which varyin nucleotide sequence by redundancy of the genetic code. The terms“polynucleotide variant” and “variant” also include naturally occurringallelic variants.

Nucleic Acid Constructs

The present invention includes nucleic acid constructs comprising thepolynucleotides of the invention, and vectors and host cells containingthese, methods of their production and use, and uses thereof. Thepresent invention refers to elements which are operably connected orlinked. “Operably connected” or “operably linked” and the like refer toa linkage of polynucleotide elements in a functional relationship.Typically, operably connected nucleic acid sequences are contiguouslylinked and, where necessary to join two protein coding regions,contiguous and in reading frame. A coding sequence is “operablyconnected to” another coding sequence when RNA polymerase willtranscribe the two coding sequences into a single RNA, which iftranslated is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein, the term “cis-acting sequence”, “cis-acting element” or“cis-regulatory region” or “regulatory region” or similar term shall betaken to mean any sequence of nucleotides, which when positionedappropriately and connected relative to an expressible genetic sequence,is capable of regulating, at least in part, the expression of thegenetic sequence. Those skilled in the art will be aware that acis-regulatory region may be capable of activating, silencing,enhancing, repressing or otherwise altering the level of expressionand/or cell-type-specificity and/or developmental specificity of a genesequence at the transcriptional or post-transcriptional level. Inpreferred embodiments of the present invention, the cis-acting sequenceis an activator sequence that enhances or stimulates the expression ofan expressible genetic sequence.

“Operably connecting” a promoter or enhancer element to a transcribablepolynucleotide means placing the transcribable polynucleotide (e.g.,protein-encoding polynucleotide or other transcript) under theregulatory control of a promoter, which then controls the transcriptionof that polynucleotide. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition a promoter or variant thereof at a distance from thetranscription start site of the transcribable polynucleotide which isapproximately the same as the distance between that promoter and theprotein coding region it controls in its natural setting; i.e., the genefrom which the promoter is derived. As is known in the art, somevariation in this distance can be accommodated without loss of function.Similarly, the preferred positioning of a regulatory sequence element(e.g., an operator, enhancer etc) with respect to a transcribablepolynucleotide to be placed under its control is defined by thepositioning of the element in its natural setting; i.e., the genes fromwhich it is derived.

“Promoter” or “promoter sequence” as used herein refers to a region of agene, generally upstream (5′) of the RNA encoding region, which controlsthe initiation and level of transcription in the cell of interest. A“promoter” includes the transcriptional regulatory sequences of aclassical genomic gene, such as a TATA box and CCAAT box sequences, aswell as additional regulatory elements (i.e., upstream activatingsequences, enhancers and silencers) that alter gene expression inresponse to developmental and/or environmental stimuli, or in atissue-specific or cell-type-specific manner. A promoter is usually, butnot necessarily (for example, some PolIII promoters), positionedupstream of a structural gene, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.Promoters may contain additional specific regulatory elements, locatedmore distal to the start site to further enhance expression in a cell,and/or to alter the timing or inducibility of expression of a structuralgene to which it is operably connected.

“Constitutive promoter” refers to a promoter that directs expression ofan operably linked transcribed sequence in many or all tissues of anorganism such as a plant. The term constitutive as used herein does notnecessarily indicate that a gene is expressed at the same level in allcell types, but that the gene is expressed in a wide range of celltypes, although some variation in level is often detectable. “Selectiveexpression” as used herein refers to expression almost exclusively inspecific organs of, for example, the plant, such as, for example,endosperm, embryo, leaves, fruit, tubers or root. In a preferredembodiment, a promoter is expressed selectively or preferentially inleaves and/or stems of a plant, preferably a cereal plant. Selectiveexpression may therefore be contrasted with constitutive expression,which refers to expression in many or all tissues of a plant under mostor all of the conditions experienced by the plant.

Selective expression may also result in compartmentation of the productsof gene expression in specific plant tissues, organs or developmentalstages. Compartmentation in specific subcellular locations such as theplastid, cytosol, vacuole, or apoplastic space may be achieved by theinclusion in the structure of the gene product of appropriate signals,eg. a signal peptide, for transport to the required cellularcompartment, or in the case of the semi-autonomous organelles (plastidsand mitochondria) by integration of the transgene with appropriateregulatory sequences directly into the organelle genome.

A “tissue-specific promoter” or “organ-specific promoter” is a promoterthat is preferentially expressed in one tissue or organ relative to manyother tissues or organs, preferably most if not all other tissues ororgans in, for example, a plant. Typically, the promoter is expressed ata level 10-fold higher in the specific tissue or organ than in othertissues or organs.

In an embodiment, the promoter is a stem-specific promoter, aleaf-specific promoter or a promoter which directs gene expression in anaerial part of the plant (at least stems and leaves) (green tissuespecific promoter) such as a ribulose-1,5-bisphosphate carboxylaseoxygenase (RUBISCO) promoter.

Examples of stem-specific promoters include, but are not limited tothose described in U.S. Pat. No. 5,625,136, and Bam et al. (2008).

The promoters contemplated by the present invention may be native to thehost plant to be transformed or may be derived from an alternativesource, where the region is functional in the host plant. Other sourcesinclude the Agrobacterium T-DNA genes, such as the promoters of genesfor the biosynthesis of nopaline, octapine, mannopine, or other opinepromoters, tissue specific promoters (see, e.g., U.S. Pat. No. 5,459,252and WO 91/13992); promoters from viruses (including host specificviruses), or partially or wholly synthetic promoters. Numerous promotersthat are functional in mono- and dicotyledonous plants are well known inthe art (see, for example, Greve, 1983; Salomon et al., 1984; Garfinkelet al., 1983; Barker et al., 1983); including various promoters isolatedfrom plants and viruses such as the cauliflower mosaic virus promoter(CaMV 35S, 19S). Non-limiting methods for assessing promoter activityare disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989,supra) and U.S. Pat. No. 5,164,316.

Alternatively or additionally, the promoter may be an inducible promoteror a developmentally regulated promoter which is capable of drivingexpression of the introduced polynucleotide at an appropriatedevelopmental stage of the, for example, plant. Other cis-actingsequences which may be employed include transcriptional and/ortranslational enhancers. Enhancer regions are well known to personsskilled in the art, and can include an ATG translational initiationcodon and adjacent sequences. When included, the initiation codon shouldbe in phase with the reading frame of the coding sequence relating tothe foreign or exogenous polynucleotide to ensure translation of theentire sequence if it is to be translated. Translational initiationregions may be provided from the source of the transcriptionalinitiation region, or from a foreign or exogenous polynucleotide. Thesequence can also be derived from the source of the promoter selected todrive transcription, and can be specifically modified so as to increasetranslation of the mRNA.

The nucleic acid construct of the present invention may comprise a 3′non-translated sequence from about 50 to 1,000 nucleotide base pairswhich may include a transcription termination sequence. A 3′non-translated sequence may contain a transcription termination signalwhich may or may not include a polyadenylation signal and any otherregulatory signals capable of effecting mRNA processing. Apolyadenylation signal functions for addition of polyadenylic acidtracts to the 3′ end of a mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon. Transcriptiontermination sequences which do not include a polyadenylation signalinclude terminators for Poll or PolIII RNA polymerase which comprise arun of four or more thymidines. Examples of suitable 3′ non-translatedsequences are the 3′ transcribed non-translated regions containing apolyadenylation signal from an octopine synthase (ocs) gene or nopalinesynthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983).Suitable 3′ non-translated sequences may also be derived from plantgenes such as the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO)gene, although other 3′ elements known to those of skill in the art canalso be employed.

As the DNA sequence inserted between the transcription initiation siteand the start of the coding sequence, i.e., the untranslated 5′ leadersequence (5′UTR), can influence gene expression if it is translated aswell as transcribed, one can also employ a particular leader sequence.Suitable leader sequences include those that comprise sequences selectedto direct optimum expression of the foreign or endogenous DNA sequence.For example, such leader sequences include a preferred consensussequence which can increase or maintain mRNA stability and preventinappropriate initiation of translation as for example described byJoshi (1987).

Vectors

The present invention includes use of vectors for manipulation ortransfer of genetic constructs. By “chimeric vector” is meant a nucleicacid molecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, or plant virus, into which a nucleic acidsequence may be inserted or cloned. A vector preferably isdouble-stranded DNA and contains one or more unique restriction sitesand may be capable of autonomous replication in a defined host cellincluding a target cell or tissue or a progenitor cell or tissuethereof, or capable of integration into the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectormay be an autonomously replicating vector, i.e., a vector that exists asan extrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into a cell,is integrated into the genome of the recipient cell and replicatedtogether with the chromosome(s) into which it has been integrated. Avector system may comprise a single vector or plasmid, two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the cell into which the vector is to be introduced. The vector mayalso include a selection marker such as an antibiotic resistance gene, aherbicide resistance gene or other gene that can be used for selectionof suitable transformants. Examples of such genes are well known tothose of skill in the art.

The nucleic acid construct of the invention can be introduced into avector, such as a plasmid. Plasmid vectors typically include additionalnucleic acid sequences that provide for easy selection, amplification,and transformation of the expression cassette in prokaryotic andeukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, orbinary vectors containing one or more T-DNA regions. Additional nucleicacid sequences include origins of replication to provide for autonomousreplication of the vector, selectable marker genes, preferably encodingantibiotic or herbicide resistance, unique multiple cloning sitesproviding for multiple sites to insert nucleic acid sequences or genesencoded in the nucleic acid construct, and sequences that enhancetransformation of prokaryotic and eukaryotic (especially plant) cells.

By “marker gene” is meant a gene that imparts a distinct phenotype tocells expressing the marker gene and thus allows such transformed cellsto be distinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. The actualchoice of a marker is not crucial as long as it is functional (i.e.,selective) in combination with the plant cells of choice. The markergene and the foreign or exogenous polynucleotide of interest do not haveto be linked, since co-transformation of unlinked genes as, for example,described in U.S. Pat. No. 4,399,216 is also an efficient process inplant transformation.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, erythromycin, chloramphenicolor tetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as, forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as, for example, described in WO 87/05327, anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al. (1988), a bar gene conferringresistance against bialaphos as, for example, described in WO91/02071; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal., 1988); a mutant acetolactate synthase gene (ALS), which confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (EP 154,204); a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan; or a dalapon dehalogenasegene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known, an aequorin gene(Prasher et al., 1985), which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al.,1986), which allows for bioluminescence detection, and others known inthe art. By “reporter molecule” as used in the present specification ismeant a molecule that, by its chemical nature, provides an analyticallyidentifiable signal that facilitates determination of promoter activityby reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into thegenome of, for example, the plant. Accordingly, the nucleic acidcomprises appropriate elements which allow the molecule to beincorporated into the genome, or the construct is placed in anappropriate vector which can be incorporated into a chromosome of aplant cell.

One embodiment of the present invention includes a recombinant vector,which includes at least one polynucleotide molecule of the presentinvention, inserted into any vector capable of delivering the nucleicacid molecule into a host cell. Such a vector contains heterologousnucleic acid sequences, that is nucleic acid sequences that are notnaturally found adjacent to nucleic acid molecules of the presentinvention and that preferably are derived from a species other than thespecies from which the nucleic acid molecule(s) are derived. The vectorcan be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

The level of a protein of the invention may be modulated by increasingthe level of expression of a nucleotide sequence that codes for theprotein in a plant cell, or decreasing the level of expression of a geneencoding the protein in the plant, leading to modified pathogenresistance. The level of expression of a gene may be modulated byaltering the copy number per cell, for example by introducing asynthetic genetic construct comprising the coding sequence and atranscriptional control element that is operably connected thereto andthat is functional in the cell. A plurality of transformants may beselected and screened for those with a favourable level and/orspecificity of transgene expression arising from influences ofendogenous sequences in the vicinity of the transgene integration site.A favourable level and pattern of transgene expression is one whichresults in a substantial modification of pathogen resistance or otherphenotype. Alternatively, a population of mutagenized seed or apopulation of plants from a breeding program may be screened forindividual lines with altered pathogen resistance or other phenotypeassociated with pathogen resistance.

Recombinant Cells

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention, or progeny cells thereof.Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. A recombinant cell may remainunicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained. Preferred hostcells are plant cells, more preferably cells of a cereal plant, morepreferably barley or wheat cells, and even more preferably a wheat cell.

Transgenic Plants

The term “plant” as used herein as a noun refers to whole plants andrefers to any member of the Kingdom Plantae, but as used as an adjectiverefers to any substance which is present in, obtained from, derivedfrom, or related to a plant, such as for example, plant organs (e.g.leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plantcells and the like. Plantlets and germinated seeds from which roots andshoots have emerged are also included within the meaning of “plant”. Theterm “plant parts” as used herein refers to one or more plant tissues ororgans which are obtained from a plant and which comprises genomic DNAof the plant. Plant parts include vegetative structures (for example,leaves, stems), roots, floral organs/structures, seed (including embryo,cotyledons, and seed coat), plant tissue (for example, vascular tissue,ground tissue, and the like), cells and progeny of the same. The term“plant cell” as used herein refers to a cell obtained from a plant or ina plant and includes protoplasts or other cells derived from plants,gamete-producing cells, and cells which regenerate into whole plants.Plant cells may be cells in culture. By “plant tissue” is meantdifferentiated tissue in a plant or obtained from a plant (“explant”) orundifferentiated tissue derived from immature or mature embryos, seeds,roots, shoots, fruits, tubers, pollen, tumor tissue, such as crowngalls, and various forms of aggregations of plant cells in culture, suchas calli. Exemplary plant tissues in or from seeds are cotyledon, embryoand embryo axis. The invention accordingly includes plants and plantparts and products comprising these.

As used herein, the term “seed” refers to “mature seed” of a plant,which is either ready for harvesting or has been harvested from theplant, such as is typically harvested commercially in the field, or as“developing seed” which occurs in a plant after fertilisation and priorto seed dormancy being established and before harvest.

A “transgenic plant” as used herein refers to a plant that contains anucleic acid construct not found in a wild-type plant of the samespecies, variety or cultivar. That is, transgenic plants (transformedplants) contain genetic material (a transgene) that they did not containprior to the transformation. The transgene may include genetic sequencesobtained from or derived from a plant cell, or another plant cell, or anon-plant source, or a synthetic sequence. Typically, the transgene hasbeen introduced into the plant by human manipulation such as, forexample, by transformation but any method can be used as one of skill inthe art recognizes. The genetic material is preferably stably integratedinto the genome of the plant. The introduced genetic material maycomprise sequences that naturally occur in the same species but in arearranged order or in a different arrangement of elements, for examplean antisense sequence. Plants containing such sequences are includedherein in “transgenic plants”.

A “non-transgenic plant” is one which has not been genetically modifiedby the introduction of genetic material by recombinant DNA techniques.In a preferred embodiment, the transgenic plants are homozygous for eachand every gene that has been introduced (transgene) so that theirprogeny do not segregate for the desired phenotype.

As used herein, the term “compared to an isogenic plant”, or similarphrases, refers to a plant which is isogenic relative to the transgenicplant but without the transgene of interest. Preferably, thecorresponding non-transgenic plant is of the same cultivar or variety asthe progenitor of the transgenic plant of interest, or a sibling plantline which lacks the construct, often termed a “segregant”, or a plantof the same cultivar or variety transformed with an “empty vector”construct, and may be a non-transgenic plant. “Wild type”, as usedherein, refers to a cell, tissue or plant that has not been modifiedaccording to the invention. Wild-type cells, tissue or plants may beused as controls to compare levels of expression of an exogenous nucleicacid or the extent and nature of trait modification with cells, tissueor plants modified as described herein.

Transgenic plants, as defined in the context of the present inventioninclude progeny of the plants which have been genetically modified usingrecombinant techniques, wherein the progeny comprise the transgene ofinterest. Such progeny may be obtained by self-fertilisation of theprimary transgenic plant or by crossing such plants with another plantof the same species. This would generally be to modulate the productionof at least one protein defined herein in the desired plant or plantorgan. Transgenic plant parts include all parts and cells of said plantscomprising the transgene such as, for example, cultured tissues, callusand protoplasts.

Plants contemplated for use in the practice of the present inventioninclude both monocotyledons and dicotyledons. Target plants include, butare not limited to, the following: cereals (for example, wheat, barley,rye, oats, rice, maize, sorghum and related crops); grapes; beet (sugarbeet and fodder beet); pomes, stone fruit and soft fruit (apples, pears,plums, peaches, almonds, cherries, strawberries, raspberries andblack-berries); leguminous plants (beans, lentils, peas, soybeans); oilplants (rape or other Brassicas, mustard, poppy, olives, sunflowers,safflower, flax, coconut, castor oil plants, cocoa beans, groundnuts);cucumber plants (marrows, cucumbers, melons); fibre plants (cotton,flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit,mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots,onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon,camphor); or plants such as maize, tobacco, nuts, coffee, sugar cane,tea, vines, hops, turf, bananas and natural rubber plants, as well asornamentals (flowers, shrubs, broad-leaved trees and evergreens, such asconifers). Preferably, the plant is a cereal plant, more preferablywheat, rice, maize, triticale, oats or barley, even more preferablywheat.

As used herein, the term “wheat” refers to any species of the GenusTriticum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. Wheat includes “hexaploid wheat”which has genome organization of AABBDD, comprised of 42 chromosomes,and “tetraploid wheat” which has genome organization of AABB, comprisedof 28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta, T.macha, T. compactum, T sphaerococcum, T. vavilovii, and interspeciescross thereof. A preferred species of hexaploid wheat is T. aestivum sspaestivum (also termed “breadwheat”). Tetraploid wheat includes T. durum(also referred to herein as durum wheat or Triticum turgidum ssp.durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspeciescross thereof. In addition, the term “wheat” includes potentialprogenitors of hexaploid or tetraploid Triticum sp. such as T. uartu, T.monococcum or T. boeoticum for the A genome, Aegilops speltoides for theB genome, and T. tauschii (also known as Aegilops squarrosa or Aegilopstauschii) for the D genome. Particularly preferred progenitors are thoseof the A genome, even more preferably the A genome progenitor is T.monococcum. A wheat cultivar for use in the present invention may belongto, but is not limited to, any of the above-listed species. Alsoencompassed are plants that are produced by conventional techniquesusing Triticum sp. as a parent in a sexual cross with a non-Triticumspecies (such as rye [Secale cereale]), including but not limited toTriticale.

As used herein, the term “barley” refers to any species of the GenusHordeum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. It is preferred that the plantis of a Hordeum species which is commercially cultivated such as, forexample, a strain or cultivar or variety of Hordeum vulgare or suitablefor commercial production of grain.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide of thepresent invention in the desired plant or plant organ. Transgenic plantscan be produced using techniques known in the art, such as thosegenerally described in A. Slater et al., Plant Biotechnology—The GeneticManipulation of Plants, Oxford University Press (2003), and P. Christouand H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons(2004).

In a preferred embodiment, the transgenic plants are homozygous for eachand every gene that has been introduced (transgene) so that theirprogeny do not segregate for the desired phenotype. The transgenicplants may also be heterozygous for the introduced transgene(s), suchas, for example, in F1 progeny which have been grown from hybrid seed.Such plants may provide advantages such as hybrid vigour, well known inthe art.

As used herein, the “other genetic markers” may be any molecules whichare linked to a desired trait of a plant. Such markers are well known tothose skilled in the art and include molecular markers linked to genesdetermining traits such disease resistance, yield, plant morphology,grain quality, dormancy traits, grain colour, gibberellic acid contentin the seed, plant height, flour colour and the like. Examples of suchgenes are the stripe rust resistance genes Yr10 or Yr17, the nematoderesistance genes such as Cre1 and Cre3, alleles at glutenin loci thatdetermine dough strength such as Ax, Bx, Dx, Ay, By and Dy alleles, theRht genes that determine a semi-dwarf growth habit and therefore lodgingresistance.

Four general methods for direct delivery of a gene into cells have beendescribed: (1) chemical methods (Graham et al., 1973); (2) physicalmethods such as microinjection (Capecchi, 1980); electroporation (see,for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat.No. 4,945,050 and U.S. Pat. No. 5,141,131); (3) viral vectors (Clapp,1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediatedmechanisms (Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. A particle delivery system suitable for use with the presentinvention is the helium acceleration PDS-1000/He gun is available fromBio-Rad Laboratories. For the bombardment, immature embryos or derivedtarget cells such as scutella or calli from immature embryos may bearranged on solid culture medium.

In another alternative embodiment, plastids can be stably transformed.Method disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S.Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,104,310, U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135).Further, the integration of the T-DNA is a relatively precise processresulting in few rearrangements. The region of DNA to be transferred isdefined by the border sequences, and intervening DNA is usually insertedinto the plant genome.

Agrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations asdescribed (Klee et al., Plant DNA Infectious Agents, Hohn and Schell,(editors), Springer-Verlag, New York, (1985): 179-203). Moreover,technological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described have convenientmulti-linker regions flanked by a promoter and a polyadenylation sitefor direct expression of inserted polypeptide coding genes and aresuitable for present purposes. In addition, Agrobacterium containingboth armed and disarmed Ti genes can be used for the transformations. Inthose plant varieties where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, Breeding Methods for Cultivar Development, J.Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., Methods for Plant MolecularBiology, Academic Press, San Diego, (1988)). This regeneration andgrowth process typically includes the steps of selection of transformedcells, culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011);Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., 1996); and pea(Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447, WO97/048814, U.S. Pat. No. 5,589,617, U.S. Pat. No. 6,541,257, and othermethods are set out in WO 99/14314. Preferably, transgenic wheat orbarley plants are produced by Agrobacterium tumefaciens mediatedtransformation procedures. Vectors carrying the desired nucleic acidconstruct may be introduced into regenerable wheat cells of tissuecultured plants or explants, or suitable plant systems such asprotoplasts. The regenerable wheat cells are preferably from thescutellum of immature embryos, mature embryos, callus derived fromthese, or the meristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

Marker Assisted Selection

Marker assisted selection is a well recognised method of selecting forheterozygous plants required when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants. To further speed up the backcrossingprogram, the embryo from immature seeds (25 days post anthesis) may beexcised and grown up on nutrient media under sterile conditions, ratherthan allowing full seed maturity. This process, termed “embryo rescue”,used in combination with DNA extraction at the three leaf stage andanalysis of at least one Lr67 allele or variant that confers upon theplant resistance to one or more biotrophic fungal pathogen(s),preferably to one or more or all of leaf rust, stripe rust, stem rustand powdery mildew, allows rapid selection of plants carrying thedesired trait, which may be nurtured to maturity in the greenhouse orfield for subsequent further backcrossing to the recurrent parent.

Any molecular biological technique known in the art can be used in themethods of the present invention. Such methods include, but are notlimited to, the use of nucleic acid amplification, nucleic acidsequencing, nucleic acid hybridization with suitably labeled probes,single-strand conformational analysis (SSCA), denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavageanalysis (CCM), catalytic nucleic acid cleavage or a combination thereof(see, for example, Lemieux, 2000; Langridge et al., 2001). The inventionalso includes the use of molecular marker techniques to detectpolymorphisms linked to alleles of the (for example) Lr67 gene whichconfers upon the plant resistance to one or more biotrophic fungalpathogen(s), preferably to one or more or all of leaf rust, stripe rust,stem rust and powdery mildew. Such methods include the detection oranalysis of restriction fragment length polymorphisms (RFLP), RAPD,amplified fragment length polymorphisms (AFLP) and microsatellite(simple sequence repeat, SSR) polymorphisms. The closely linked markerscan be obtained readily by methods well known in the art, such as BulkedSegregant Analysis, as reviewed by Langridge et al., (2001).

In an embodiment, a linked loci for marker assisted selection is atleast within 1 cM, or 0.5 cM, or 0.1 cM, or 0.01 cM from a gene encodinga polypeptide of the invention.

The “polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are known in theart, and are taught, for example, in “PCR” (M. J. McPherson and S. GMoller (editors), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCRcan be performed on cDNA obtained from reverse transcribing mRNAisolated from plant cells expressing a Lr67 gene or allele which confersupon the plant resistance to one or more biotrophic fungal pathogen(s),preferably to one or more or all of leaf rust, stripe rust, stem rustand powdery mildew. However, it will generally be easier if PCR isperformed on genomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridisingin a sequence specific fashion to the target sequence and being extendedduring the PCR. Amplicons or PCR products or PCR fragments oramplification products are extension products that comprise the primerand the newly synthesized copies of the target sequences. Multiplex PCRsystems contain multiple sets of primers that result in simultaneousproduction of more than one amplicon. Primers may be perfectly matchedto the target sequence or they may contain internal mismatched basesthat can result in the introduction of restriction enzyme or catalyticnucleic acid recognition/cleavage sites in specific target sequences.Primers may also contain additional sequences and/or contain modified orlabelled nucleotides to facilitate capture or detection of amplicons.Repeated cycles of heat denaturation of the DNA, annealing of primers totheir complementary sequences and extension of the annealed primers withpolymerase result in exponential amplification of the target sequence.The terms target or target sequence or template refer to nucleic acidsequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.,(supra) and Sambrook et al., (supra). Sequencing can be carried out byany suitable method, for example, dideoxy sequencing, chemicalsequencing or variations thereof. Direct sequencing has the advantage ofdetermining variation in any base pair of a particular sequence.

TILLING

Plants of the invention can be produced using the process known asTILLING (Targeting Induced Local Lesions IN Genomes). In a first step,introduced mutations such as novel single base pair changes are inducedin a population of plants by treating seeds (or pollen) with a chemicalmutagen, and then advancing plants to a generation where mutations willbe stably inherited. DNA is extracted, and seeds are stored from allmembers of the population to create a resource that can be accessedrepeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cel I, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique.

TILLING is further described in Slade and Knauf (2005), and Henikoff etal. (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Plant/Grain Processing

Grain/seed of the invention, preferably cereal grain and more preferablywheat grain, or other plant parts of the invention, can be processed toproduce a food ingredient, food or non-food product using any techniqueknown in the art.

In one embodiment, the product is whole grain flour such as, forexample, an ultrafine-milled whole grain flour, or a flour made fromabout 100% of the grain. The whole grain flour includes a refined flourconstituent (refined flour or refined flour) and a coarse fraction (anultrafine-milled coarse fraction).

Refined flour may be flour which is prepared, for example, by grindingand bolting cleaned grain such as wheat or barley grain. The particlesize of refined flour is described as flour in which not less than 98%passes through a cloth having openings not larger than those of wovenwire cloth designated “212 micrometers (U.S. Wire 70)”. The coarsefraction includes at least one of: bran and germ. For instance, the germis an embryonic plant found within the grain kernel. The germ includeslipids, fiber, vitamins, protein, minerals and phytonutrients, such asflavonoids. The bran includes several cell layers and has a significantamount of lipids, fiber, vitamins, protein, minerals and phytonutrients,such as flavonoids. Further, the coarse fraction may include an aleuronelayer which also includes lipids, fiber, vitamins, protein, minerals andphytonutrients, such as flavonoids. The aleurone layer, whiletechnically considered part of the endosperm, exhibits many of the samecharacteristics as the bran and therefore is typically removed with thebran and germ during the milling process. The aleurone layer containsproteins, vitamins and phytonutrients, such as ferulic acid.

Further, the coarse fraction may be blended with the refined flourconstituent. The coarse fraction may be mixed with the refined flourconstituent to form the whole grain flour, thus providing a whole grainflour with increased nutritional value, fiber content, and antioxidantcapacity as compared to refined flour. For example, the coarse fractionor whole grain flour may be used in various amounts to replace refinedor whole grain flour in baked goods, snack products, and food products.The whole grain flour of the present invention (i.e.-ultrafine-milledwhole grain flour) may also be marketed directly to consumers for use intheir homemade baked products. In an exemplary embodiment, a granulationprofile of the whole grain flour is such that 98% of particles by weightof the whole grain flour are less than 212 micrometers.

In further embodiments, enzymes found within the bran and germ of thewhole grain flour and/or coarse fraction are inactivated in order tostabilize the whole grain flour and/or coarse fraction. Stabilization isa process that uses steam, heat, radiation, or other treatments toinactivate the enzymes found in the bran and germ layer. Flour that hasbeen stabilized retains its cooking characteristics and has a longershelf life.

In additional embodiments, the whole grain flour, the coarse fraction,or the refined flour may be a component (ingredient) of a food productand may be used to product a food product. For example, the food productmay be a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, anEnglish muffin, a muffin, a pita bread, a quickbread, arefrigerated/frozen dough product, dough, baked beans, a burrito, chili,a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, a readyto eat meal, stuffing, a microwaveable meal, a brownie, a cake, acheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll,a candy bar, a pie crust, pie filling, baby food, a baking mix, abatter, a breading, a gravy mix, a meat extender, a meat substitute, aseasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup,sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, lo meinnoodles, an ice cream inclusion, an ice cream bar, an ice cream cone, anice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, anextruded snack, a fruit and grain bar, a microwaveable snack product, anutritional bar, a pancake, a par-baked bakery product, a pretzel, apudding, a granola-based product, a snack chip, a snack food, a snackmix, a waffle, a pizza crust, animal food or pet food.

In alternative embodiments, the whole grain flour, refined flour, orcoarse fraction may be a component of a nutritional supplement. Forinstance, the nutritional supplement may be a product that is added tothe diet containing one or more additional ingredients, typicallyincluding: vitamins, minerals, herbs, amino acids, enzymes,antioxidants, herbs, spices, probiotics, extracts, prebiotics and fiber.The whole grain flour, refined flour or coarse fraction of the presentinvention includes vitamins, minerals, amino acids, enzymes, and fiber.For instance, the coarse fraction contains a concentrated amount ofdietary fiber as well as other essential nutrients, such as B-vitamins,selenium, chromium, manganese, magnesium, and antioxidants, which areessential for a healthy diet. For example 22 grams of the coarsefraction of the present invention delivers 33% of an individual's dailyrecommend consumption of fiber. The nutritional supplement may includeany known nutritional ingredients that will aid in the overall health ofan individual, examples include but are not limited to vitamins,minerals, other fiber components, fatty acids, antioxidants, aminoacids, peptides, proteins, lutein, ribose, omega-3 fatty acids, and/orother nutritional ingredients. The supplement may be delivered in, butis not limited to the following forms: instant beverage mixes,ready-to-drink beverages, nutritional bars, wafers, cookies, crackers,gel shots, capsules, chews, chewable tablets, and pills. One embodimentdelivers the fiber supplement in the form of a flavored shake or malttype beverage, this embodiment may be particularly attractive as a fibersupplement for children.

In an additional embodiment, a milling process may be used to make amulti-grain flour or a multi-grain coarse fraction. For example, branand germ from one type of grain may be ground and blended with groundendosperm or whole grain cereal flour of another type of cereal.Alternatively bran and germ of one type of grain may be ground andblended with ground endosperm or whole grain flour of another type ofgrain. It is contemplated that the present invention encompasses mixingany combination of one or more of bran, germ, endosperm, and whole grainflour of one or more grains. This multi-grain approach may be used tomake custom flour and capitalize on the qualities and nutritionalcontents of multiple types of cereal grains to make one flour.

It is contemplated that the whole grain flour, coarse fraction and/orgrain products of the present invention may be produced by any millingprocess known in the art. An exemplary embodiment involves grindinggrain in a single stream without separating endosperm, bran, and germ ofthe grain into separate streams. Clean and tempered grain is conveyed toa first passage grinder, such as a hammermill, roller mill, pin mill,impact mill, disc mill, air attrition mill, gap mill, or the like. Aftergrinding, the grain is discharged and conveyed to a sifter. Further, itis contemplated that the whole grain flour, coarse fraction and/or grainproducts of the present invention may be modified or enhanced by way ofnumerous other processes such as: fermentation, instantizing, extrusion,encapsulation, toasting, roasting, or the like.

Malting

A malt-based beverage provided by the present invention involves alcoholbeverages (including distilled beverages) and non-alcohol beverages thatare produced by using malt as a part or whole of their startingmaterial. Examples include beer, happoshu (low-malt beer beverage),whisky, low-alcohol malt-based beverages (e.g., malt-based beveragescontaining less than 1% of alcohols), and non-alcohol beverages.

Malting is a process of controlled steeping and germination followed bydrying of the grain such as barley and wheat grain. This sequence ofevents is important for the synthesis of numerous enzymes that causegrain modification, a process that principally depolymerizes the deadendosperm cell walls and mobilizes the grain nutrients. In thesubsequent drying process, flavour and colour are produced due tochemical browning reactions. Although the primary use of malt is forbeverage production, it can also be utilized in other industrialprocesses, for example as an enzyme source in the baking industry, or asa flavouring and colouring agent in the food industry, for example asmalt or as a malt flour, or indirectly as a malt syrup, etc.

In one embodiment, the present invention relates to methods of producinga malt composition. The method preferably comprises the steps of:

(i) providing grain, such as barley or wheat grain, of the invention,

(ii) steeping said grain,

(iii) germinating the steeped grains under predetermined conditions and

(iv) drying said germinated grains.

For example, the malt may be produced by any of the methods described inHoseney (Principles of Cereal Science and Technology, Second Edition,1994: American Association of Cereal Chemists, St. Paul, Minn.).However, any other suitable method for producing malt may also be usedwith the present invention, such as methods for production of specialitymalts, including, but limited to, methods of roasting the malt.

Malt is mainly used for brewing beer, but also for the production ofdistilled spirits. Brewing comprises wort production, main and secondaryfermentations and post-treatment. First the malt is milled, stirred intowater and heated. During this “mashing”, the enzymes activated in themalting degrade the starch of the kernel into fermentable sugars. Theproduced wort is clarified, yeast is added, the mixture is fermented anda post-treatment is performed.

EXAMPLES Example 1. The Lr67/Yr46 Gene is an Adult Plant Resistance GeneDistinct from Lr34/Yr18 and Lr46/Yr29 INTRODUCTION

Rust resistance genes in wheat are classified into two broad categorieswhich are referred to as seedling and adult plant resistance (APR)genes. Seedling resistance genes are detected phenotypically afterchallenge of the plant with the rust pathogen and are observed duringboth the seedling and adult plant stages; as such they confer aresistance phenotype during all stages of plant growth. In contrast, APRis generally not observed at the seedling stage but instead is detectedat the post-seedling stage of growth and often as resistance to thepathogen in plants growing in the field. In a few exceptions, some APRgenes can be induced to express in seedlings by varying the growthtemperature and light conditions, but are not expressed in seedlingsunder all growth conditions. Additionally, seedling resistance genestypically exhibit phenotypes of major effect and with varying infectiontypes whereas most of the APR genes are partial in effect with varyinglevels of disease severity. Race specificity is more common with theseedling resistance genes. Of the few APR genes that have been studiedin wheat, most appear to be race non-specific and a limited number areclearly race specific. Those of the race non-specific class with partialresistance are associated with a slow rusting phenotype first describedby Caldwell (1968). Typically, slow rusting resistance shows longerlatent periods, fewer uredinia and smaller uredinia sizes within thefirst two weeks post inoculation when compared to susceptible plants.

One of the well characterised race non-specific resistance genes is theadult plant leaf rust resistance gene Lr34 (WO2010/022443). Earlierreferred to as LrT2 (Dyck 1977, 1987) it is present in older SouthAmerican wheat cultivars such as Frontana and its derivatives, some ofthe early crossbred wheat cultivars at the beginning of last century andsome wheat landraces (non-cultivated varieties isolated from wild-grownwheats) in particular those of Chinese origin (Borghi 2001; Kolmer etal., 2008). An important feature of Lr34 was that virulence in the wheatleaf rust pathogen has not been reported to date, and the enhancedeffect of rust resistance when the Lr34 gene was combined in wheatcultivars with other race specific leaf rust resistance genes hascontributed to the durability of wheat cultivars with Lr34 genecombinations (Kolmer, 1996). However, variability in the extent of rustcolony development between leaf rust isolates on Lr34 has been reported(Bender and Pretorius, 2000). Co-segregation of Lr34 with the adultplant stripe rust resistance gene Yr18 (McIntosh 1992; Singh 1992) inexhibiting dual rust resistance in numerous wheat backgrounds may havecontributed to the continued widespread use of the Lr34/Yr18 germplasmin wheat breeding. Subsequent observations that the Lr34/Yr18 locus alsocontributed to partial resistance against adult plant powdery mildew(Pm38) highlighted the multi-pathogen nature of the Lr34/Yr18/Pm38 locuson the short arm of wheat chromosome 7D (Spielmeyer et al., 2005;Lillemo et al., 2008).

Chemical and physical mutagenesis were used to investigate themulti-pathogen resistance locus containing Lr34 on wheat chromosome 7DS(Spielmeyer et al., 2008). Susceptible mutants were recovered for whichthere was no loss of DNA markers in the QTL interval on 7DS. Thesemutants were subsequently shown to be point mutations of which thechemical mutagen had created single base substitutions. Additionalmutants generated by gamma irradiation had single base deletions withina gene encoding an ATP Binding Cassette (ABC) transporter at themulti-pathogen resistance locus (Krattinger et al., 2009). In additionto the ABC transporter, six other genes co-segregated with theresistance locus and were genetically and physically closely linked.However none of the mutants (eight independent mutants) had changes inthe additional genes. Thus mutagenic changes to the ABC transporteralone were adequate to confer loss of all three of the leaf rust, striperust and powdery mildew resistances encoded by Lr34/Yr18/Pm38. Togetherwith haplotype analysis and high resolution mapping, it was establishedthat a single gene, an ABC transporter, conferred all three resistances(Krattinger et al., 2009).

Lr67/Yr46 is Distinct from Lr34/Yr18 and Lr46/Yr29

In the course of developing near isogenic lines for leaf rust resistancein the cultivar Thatcher, Dyck (1987) observed a phenotypic spectrum inline RL6077 (Thatcher*6/PI250413), that was similar to RL6058, anear-isogenic line carrying Lr34/Yr18. In subsequent studies, the APR inRL6077 segregated independently of Lr34/Yr18 and there was evidence fora translocation difference between the lines. As a result Dyck et al.(1994) inferred that RL6077 was a carrier of the Lr34/Yr18 gene, but ona different chromosome to 7DS. With the development of closely linkedgenetic markers, and ultimately the cloning of Lr34/Yr18, it becameclear that RL6077 lacked the Lr34/Yr18a resistance haplotype present inRL6058 and therefore contained a different APR gene (Kolmer et al.,2008; Lagudah et al., 2009).

Studies on mapping populations from crosses involving RL6077 using P.triticina and P. striiformis isolates from multiple locations in Canada,Mexico and Australia, established co-segregation of the respective APRson chromosome 4DL (long arm of chromosome 4D, on the D genome ofbreadwheat) (Herrera-Foessel et al., 2011; Hiebert et al., 2010). TheseAPR genes at the resistance locus on 4DL have been designated Lr67/Yr46.

Example 2. Cloning of the Lr67 (syn=Yr46=Sr55=Pm46) Gene

In addition to the wheat genotype RL6077, two other wheat genotypes,NP876 and Sujata have been postulated to carry Lr67. This was based onthe presence of the leaf tip necrosis phenotype (Ltn), slow rusting toleaf rust infection and the simple sequence repeat (SSR) marker allelelinked to Lr67 in RL6077 (Hererra et al., 2011). As part of the strategytowards identification of co-segregating markers and cloning of the Lr67gene, the present inventors developed recombinant inbred (RI) familiesfrom crosses involving the adult plant leaf and stripe rust susceptibleparent, Avocet. These RI families were derived from the crossesAvocet×RL6077, Avocet×Sujata and Avocet×NP876. An F2 family from thecross Thatcher×Thatcher+Lr67 (RL6077) was also made. These RI familieswere then used in genetic mapping studies and mutational experiments asfollows.

Firstly, a mutagenised population of a derived line from Avocet×RL6077fixed for the Lr67 gene was conducted using gamma irradiation with adosage of 20 krad from a ⁶⁰Co source. Secondly, chemical mutagenesisusing on ethyl methanesulfonate (EMS) under standard mutagenesisconditions was used to generate a mutant population using the wheatgenotype RL6077. M2 progeny lines were produced from the mutagenisedpopulations and each tested for loss of the resistance phenotype. Fieldevaluation of the M2 mutant progeny was carried out to identify loss ofleaf and stripe rust resistance resulting from inactivation of the Lr67gene. Putative mutants were further tested at the M3 and M4 stage toselect homozygous susceptible lines. Plants of five gamma irradiatedlines (designated γ318, γ676, γ1183, γ1239 and γ1656) and two EMS lines(designated emsSu1 and emsSu2) were identified as susceptible mutants.They all showed loss of both the leaf rust and stripe rust resistancephenotypes as well as the leaf tip necrosis phenotype, suggesting that asingle gene was responsible for the three phenotypes. The wheat genotypebackground of the EMS mutants facilitated stem rust and powdery mildewassessments, and each of the mutants exhibited susceptibility to bothdiseases in contrast to the resistance observed in their sibs andoriginal parent.

In an effort to identify DNA sequences at or closely linked to the Lr67locus, fifteen homozygous recombinant inbred lines (RILs) each fromresistant (Lr67R) and susceptible (Lr67S) phenotypes were selected andsubjected to extensive AFLP and SSR analysis. A genome complexityreduction library based on PstI endonuclease restricted genomic libraryfrom pooled DNA of the 15 resistant and homozygous lines were assessedfor SNP based markers. A comparative genomics approach was alsoconducted using sequences from a wheat BAC clone containing the SSRclosely linked to Lr67 (Hererra et al., 2011) as an anchor point toidentify orthologous genes from the rice and Brachypodium genomes (FIG.1). A colinear region between Brachypodium chromosome 1 and ricechromosome 3 was then used to develop DNA markers to amplifycorresponding sequences from wheat and Aegilops tauschii, closelyrelated to the D genome progenitor to wheat chromosome 4DL (FIG. 1).

The DNA markers generated from AFLP and SNPs from the genome complexityreduction libraries produced linked markers to Lr67, but noneco-segregated with all the fifteen homozygous resistant and susceptiblerecombinant inbred lines. However, DNA fragments isolated from differentparts of a chaperonin-encoding gene containing a Hsp70 (heat shockprotein, see FIG. 1) domain isolated from the comparative genomicsapproach was completely linked to the fifteen susceptible recombinantinbred lines and absent in the fifteen resistant lines (FIG. 2). Theextent of the Hsp70 chaperonin linkage was verified in 500 recombinantinbred lines and was confirmed to show complete association with theLr67 locus. Genomic DNA blot analysis provided additional evidence thatthe gene encoding the Hsp70 chaperonin was deleted in the Lr67 resistantparental lines, RL6077, Sujata and NP876. Given that susceptible mutantswhich were loss-of-function had been isolated for Lr67, it was unlikelythat the deleted Hsp70 chaperonin gene in the resistatnt plants wassufficient for the Lr67 resistance phenotype. Consequently a search ofall predicted gene sequences in the close vicinity (within 21 kb) ofHsp70 was performed and three other genes were identified by comparativegenomics. These were genes with protein domains annotated on the rice orBrachypodium genome maps as encoding a Sec14B cytosolic factor familyprotein, a monosaccharide transporter (MST-closely related to sugartransporters-SUT) and a protein interacting/binding protein (PIP).

The nucleotide sequences of the corresponding genes in several wheatvarieties and Lr67 mutants were determined. Sequence analysis of theparental lines Thatcher (Lr67 susceptible), Avocet (Lr67 susceptible)and Thatcher+Lr67 (RL6077) showed no differences in the Sec14B genewhereas the MST and PIP genes revealed sequence polymorphisms. Two SNPs(C/G and T/G) found in the SUT gene were unique to Lr67 containingwheats (FIG. 4) while an insertion/deletion polymorphism in PIP was notdiagnostic for Lr67. Co-segregation of the SNPs in the SUT gene and Lr67adult plant rust was established in 520 recombinant inbred lines. Toensure that no additional gene sequences were missed or unknownrearrangements in the wheat chromosome 4DL region that harbours Lr67relative to the corresponding syntenic regions of Brachypodium(chromosome 1) and rice (chromosome 3), the equivalent regions fromAegilops tauschii (D genome progenitor) were analysed. A sequence contigof 70 kb showed the presence of the Hsp70, SUT and PIP genes locatedwithin a 21 kb fragment with no other predicted genes in the contig. Norecombinants were detected between these three genes when 1152 plantswere analysed from an F2 progeny population from theThatcher×Thatcher+Lr67 cross.

Analysis of the mutants that were inactivated for the Lr67 phenotype,revealed three of the gamma mutants were deleted for the SUT, PIP andsome of the markers identified from comparative genomics in the Lr67region (FIG. 3, deletions shown as the dashed lines). However four ofthe mutants (two EMS mutants and two from gamma irradiation, namely γ318and γ1239) retained the Lr67co-segregating SUT and PIP genes. Sequenceanalysis of the SUT gene in these mutants found single nucleotidechanges and small deletions in only the SUT gene and not in PIP. Each ofthe mutational changes in the four mutants occurred in amino acids foundin conserved regions of hexose sugar transporters (FIG. 4). Anadditional four mutants, obtained by sodium azide mutagenesis of RL6077,also showed single nucleotide changes that altered the amino acidcomposition of the SUT gene. On the basis of the co-segregation, andmutational analysis the inventors inferred that the SUT gene wassufficient to confer the Lr67 resistance phenotype. When tested againstthe full range of rust and mildew diseases, the wheat EMS mutants allshowed susceptibility to leaf rust, stripe rust, stem rust and powderymildew. Thus inactivation of a single gene, SUT, has an effect onmultiple diseases. The SUT gene was therefore identified conclusively asthe Lr67 gene.

The Lr67 gene encodes a protein with 514 amino acids (FIG. 5) predictedto contain 12 transmembrane domains. When predicted using the TMHMMServer v 2.0 and the PSIPRED v 3.3 program, the transmembrane domainswere predicted to be for amino acids: TMhelix1, 20-42; TMhelix2, 81-100;TMhelix3, 107-126; TMhelix4, 136-158; TMhelix5, 170-192; TMhelix6;202-221; TMhelix7, 282-304; TMhelix8, 319-341; TMhelix9, 348-370;TMhelix10, 380-402; TMhelix11, 423-445; and TMhelix12, 450-472. Each ofthe following amino acid regions: the N-terminal 19 amino acids, theamino acids between TMhelices 2 and 3, the amino acids between TMhelices 4 and 5, the amino acids between TM helices 6 and 7, the aminoacids between TMA helices 8 and 9, and the C-terminal 42 amino acidswere predicted by the same program to be located to the inside of themembrane, and the other amino acids outside of the membrane. The proteinis a member of the Major Facilitator Superfamily (MFS) which is a largeand diverse group of secondary transporters that include uniporters,symporters and antiporter proteins that facilitate transport acrosscytoplasmic or internal cell membranes of a variety of compoundsincluding sugars. Homologous polypeptides were identified by queryingthe NCBI protein database with the amino acid sequence of SEQ ID NO:1,and numerous homologs identified. The amino acid sequence of Lr67 isabout 89-93% identical to homologous polypeptides in several cerealsincluding rice, and about 80% identical to a homolog in Arabidopsis,Sugar transport protein 13 (Accession No. NP_198006).

A DNA fragment corresponding to the Lr67 gene was cloned from wheat andits nucleotide sequences compared to the cDNA sequence (SEQ ID NO:10).This revealed the presence of two introns in the protein coding regionof the gene (FIG. 14).

RT-PCR analysis showed that all three of the homoeologous Lr67 genes inthe A, B and D genomes of hexaploid wheat were expressed in the plants.The polypeptide encoded by the A genome homoeolog of the Lr67 gene was507/514 (98.6%) identical in amino acid sequence relative to SEQ IDNO:1, including having a glycine at position 144 and a valine atposition 387 which were typical of the susceptible Lr67 polypeptides.This result indicated to the inventors that the resistant Lr67polypeptide might act as a dominant-negative polypeptide, reducing theactivity of the susceptible Lr67 polypeptides encoded by the A and Bgenomes in hexaploid wheat.

The molecular basis for the differences between the polypeptides encodedby the Lr67 resistant and susceptible alleles was delimited to the twonucleotide changes that gave rise to the SNPs used in the Lr67diagnostic markers. These two nucleotide changes results in a change ofamino acids from a conserved glycine to arginine (position 144) in thepredicted fourth trans-membrane domain and a valine to leucine (position387) in the tenth trans-membrane domain (FIGS. 6, 7 and 8) withreference to SEQ ID NO:1. These two nucleotide changes were rare inwheat and were not found in the D genome progenitor or in mostcommercial wheat cultivars with the exception of the few that carryLr67. Therefore, Lr67 resistance may have originated subsequent to theformation of hexaploid wheat by hybridization from its diplod ancestoralwheats. When over a 1000 wheat landraces and accessions from a widerange of geographical sources were analysed for the two Lr67 diagnosticmarkers, the mutation that gave rise to Lr67 was very rare and localizedto a subset of landraces sourced from the Indo-Gangetic plains.

The homologs in other plant species such as Sugar transport protein 13in Arabidopsis and in cereals, without exception, do not have arginineat the position corresponding to amino acid position 144 of SEQ ID NO: 1or the leucine corresponding to amino acid position 387. Invariably, thehomologs had a glycine at the position corresponding to amino acid 144and almost always a valine at position 387 (see, for example, thealignments provided in FIGS. 9 and 10). These two amino acids weretherefore highly conserved in the SUT polypeptides, and the mutation toeither one, or both, amino acids indicative of an altered function thatis the cause of the resistance phenotype to the rust and mildewpathogens.

Example 3. Glucose Uptake Studies on Lr67 (SUT) Expressed inYeast-Variation in Polypeptide Sequence

The demonstration that the Lr67 gene encoded a protein that showedhomology to known sugar transporters led the inventors to test thefunction of the Lr67 polypeptides for sugar transport in yeast cells.

Experimental Procedure

The following experiments were completed using the protein codingregions for the Lr67 polypeptides (resistant and susceptible alleles)cloned into the yeast expression vector pRS416. This vector containedthe constitutive ADH1 promoter and CYC1 terminator for expression of theinserted coding regions. The yeast strain employed was a hexosetransport deficient variant of Saccharomyces cerevisiae namedEBY.VW4000. Uptake was determined using radio labelled [¹⁴C] glucosewith incorporation radio-assayed by liquid scintillation counting.

Results Glucose Uptake Over Time

Yeast cells transformed with the genetic constructs for expression ofeither the resistant (Lr67(res)) or susceptible (Lr67(sus)) alleles ofLr67 were incubated with 100 μM [¹⁴C] glucose for 10 minutes. Glucoseuptake was tested at 2 minute intervals. Yeast cells expressing the Lr67susceptible allele were shown to be capable of transporting glucose at ahigher rate than yeast cells expressing the Lr67 resistant allele orempty vector (FIG. 11). Indeed, the cells expressing the Lr67 resistantallele did not show detectable glucose transport activity above thecontrol, although this assay was carried out for only 10 min and was notsensitive.

Lr67 (sus) Glucose Uptake Kinetics

Concentration-dependent uptake of [¹⁴C] glucose by Lr67(sus) displayedclassical Michaelis-Menten saturation kinetics. The Lineweaver-Burkequation was used to convert data for linear regression analysis.Lr67(sus) was shown to display a high affinity for glucose, having aK_(m) of 73 μM and V_(max) of 3.02 nmol min⁻¹ g FW⁻¹ for this substrate(FIG. 12).

Amino Acid Conversion Analysis

As described above, there were two SNP differences at the nucleotidesequence level between the susceptible and resistant alleles of Lr67.This created the two amino acid substitutions in the protein productwhich include a glycine to arginine substitution at position 144 (G144R)and valine to leucine substitution at position 387 (V387L). In order totest if these amino acid substitutions, singly or together, could affectglucose uptake rates of LR67, the amino acids at positions 144 and 387in the Lr67 resistant allele were individually converted to theequivalent amino acid present in the Lr67 susceptible allele (i.e. R144Gand L387V) by mutagenesis of the cloned gene.

Yeast cells transformed with either Lr67(sus), Lr67(res), Lr67(res)R144G and Lr67(res) L387V were incubated with 100 μM [¹⁴C] glucose for10 minutes. Yeast cells expressing Lr67 (res) R144G were shown to becapable of transporting glucose at a higher rate than yeast cellsexpressing the Lr67 (res) or the Lr67 (res) L387V (FIG. 13), but not tothe full extent of Lr67(sus). This indicated that position 144 was themore important amino acid of the two substituted amino acids for rustresistance function and that the conversion of glycine to arginine(G144R) or vice versa (R144G) altered the glucose transport rate of Lr67polypeptide. However, the addition of the second amino acid substitution(L387V) also made a contribution to the glucose transport rate.

Since the deletion mutations in the Avocet×RL6077 created by gammairradiation that deleted the Lr67 gene resulted in a susceptible alleleto rust and mildew infection, the inventors concluded that the Lr67(resistant) polypeptide must have a positive function in wheat cells,certainly to be transport of a saccharide other than glucose, mostlikely to be transport of a hexose sugar. That is, conversion of theLr67 susceptible allele in wheat plants such as Avocet to the resistanceallele required the arginine at position 144 and was improved by thepresence of the leucine at position 387.

Example 4. Production of Transgenic Plants

Experiments are carried out with the cloned genes and the amino acidvariants to introduce the genetic constructs into transgenic wheat,transforming wheat plants of the cultivar Fielder using standardAgrobacterium-mediated techniques, and other plants such as cereals, toincrease the resistance to fungal pathogens such as rust and mildew.Experiments are also carried out to modify the genes encoding theArabidopsis thaliana and Vitis vinifera homologs of Lr67 to encodemutant polypeptides having the arginine at position 144 and the leucineat position 387, and to convert them into resistant polypeptides, inorder to provide resistance genes for these plant species and otherplants.

Materials and Methods

The gene encoding Lr67 was used to transform barley plants as follows. Agenomic fragment of 7133 bp was isolated from the wheat genotypeThatcher+Lr67. The fragment contained the full length genomic sequencefor the Lr67 gene and included 1318 bp of the native promoter region and1512 bp of the native terminator sequence including the 3′ untranslatedregion. The Lr67 fragment was inserted into the binary transformationvector pWBVec8. The Lr67 binary vector was transformed into the A.tumefaciens strain AGL-1 and used to produce stably transformed barleyplants (cv. Golden Promise) as described in Tingay et al. (1997).

Results

Eight independent transgenic plants were identified which weretransformed with the Lr67 transgene. All eight plants and wild-typecontrol plants which had been subjected to the tissue culture stepsinvolved in transformation but lacking the Lr67 transgene were infectedwith barley leaf rust, infecting plants of the T0 generation. One of theadvanced Lr67 transgenic lines was further tested at the T1 stage. Theleaf rust infections and subsequent culture were performed on seedlingsand mature plants of transgenic barley grown in a humidity chamber usingthe Puccinia hordei pathotype 4653P+(University of Sydney PBIC culturenumber 990492), which is avirulent on plants with resistance genes Rph3,5, 7, 10, 11, 14, and 15 and virulent on lines with resistance genesRph1, 2, 4, 6, 8, 9, 12, 13, and 19.

Rust sporulation was observed on leaves of the control plants but not onthe positive Lr67 transgenic plants of the T0 generation. All of theLr67 transgenic plants exhibited earlier leaf senescence compared withthe control plants, similar to the leaf senescence phenotype describedas leaf tip necrosis that is a characteristic feature of Lr67-mediatedresistance in wheat. When the T1 generation of plants were tested, allof the plants containing Lr67 showed the leaf rust resistance phenotypewhen tested on seedlings, whereas all of the plants lacking the Lr67gene showed leaf rust susceptibility. These results confirmed that theisolated Lr67 gene was functionally active and was sufficient to conferleaf rust resistance in barley. These experiments also extended therange of pathogen species for which Lr67 conferred resistance to includeP. hordeii in addition to Puccinia striiformis, P. triticina, P.graminis, and Blumeria vulgaris.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present applicztion claims priority from AU 2013903161 filed 21 Aug.2013, the entire contents of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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1. A recombinant cell comprising an exogenous polynucleotide encoding apolypeptide which is characterised by one or more or all of: i) whenexpressed in a plant, the polypeptide confers upon the plant resistanceto one or more biotrophic fungal pathogen(s), preferably to one or moreor all of leaf rust, stripe rust, stem rust and powdery mildew, ii) whenexpressed in a cell, the polypeptide is not as active at transportingglucose across a membrane of the cell as a polypeptide which comprisesamino acids having a sequence as provided in SEQ ID NO:4, iii) whenexpressed in a cell, the polypeptide is active as a sugar transporter,iv) the polypeptide comprises amino acids having a sequence as providedin SEQ ID NO:1 or an amino acid sequence which is at least 40% identicalto SEQ ID NO:1 or a biologically active fragment thereof, v) thepolypeptide does not comprise a glycine at a position corresponding toamino acid number 144 of SEQ ID NO:1, preferably the polypeptidecomprises an amino acid other than glycine at the position correspondingto amino acid number 144 of SEQ ID NO:1, and vi) the polypeptide doesnot comprise a valine at a position corresponding to amino acid number387 of SEQ ID NO:1, preferably the polypeptide comprises an amino acidother than valine at the position corresponding to amino acid number 387of SEQ ID NO:1, wherein the polynucleotide is operably linked to apromoter capable of directing expression of the polynucleotide in thecell.
 2. The cell of claim 1, wherein the one or more biotrophic fungalpathogen(s) is a rust or a mildew or both a rust and a mildew,preferably wherein the one or more biotrophic fungal pathogen(s) isBlumeria graminis f. sp. tritici, Fusarium graminearum, Bipolarissorokiniana, Erysiphe graminis f. sp. tritici, Puccinia graminis f. sp.tritici, Puccinia striiformis, Puccinia hordei and Puccinia recondita f.sp. tritici.
 3. The cell of claim 1 which is a plant cell.
 4. The cellof claim 3, wherein the plant cell is a cereal plant cell.
 5. The cellof claim 4, wherein the promoter directs gene expression in a leafand/or stem cell. 6.-8. (canceled)
 9. The cell of claim 1, wherein theexogenous polynucleotide is integrated into the genome of the cell. 10.The cell of claim 1, wherein the polypeptide comprises an amino acidsequence which is at least 80% identical to SEQ ID NO:1.
 11. (canceled)12. A transgenic plant comprising at least one cell of claim 1, whereinthe transgenic plant is transgenic for the exogenous polynucleotide. 13.The transgenic plant of claim 12 which has enhanced resistance to one ormore biotrophic fungal pathogen(s), preferably to a rust, a mildew orboth a rust and a mildew, more preferably to one or more or all of leafrust, stripe rust, stem rust and powdery mildew, when compared to anisogenic plant lacking the exogenous polynucleotide.
 14. The transgenicplant of claim 12 which has enhanced resistance to one or morebiotrophic fungal pathogen(s) at the seedling stage of growth.
 15. Thetransgenic plant of claim 12 which comprises one or more furtherpolynucleotides encoding a plant pathogen resistance polypeptide otherthan an Lr67 polypeptide.
 16. The transgenic plant of claim 12 which isa cereal, grape, beet or leguminous plant. 17.-19. (canceled)
 20. Aprocess for determining whether a polypeptide confers resistance orsusceptibility to one or more biotrophic fungal pathogen(s), preferablya rust, a mildew or both a rust and a mildew, more preferably to one ormore or all of leaf rust, stripe rust, stem rust and powdery mildew,comprising: i) obtaining a polynucleotide operably linked to a promoter,the polynucleotide encoding the polypeptide, wherein the polypeptidecomprises amino acids having a sequence as provided in SEQ ID NO:1 or anamino acid sequence which is at least 40% identical to one SEQ ID NO:1,or a biologically active fragment thereof, ii) introducing thepolynucleotide into a plant, iii) determining whether the level ofresistance or susceptibility to the one or more biotrophic fungalpathogen(s), preferably a rust, a mildew or both a rust and a mildew,more preferably to one or more or all of leaf rust, stripe rust, stemrust and powdery mildew, is increased or decreased relative to anisogenic plant lacking the polynucleotide, and iv) optionally, if thelevel of resistance or susceptibility is increased, selecting apolynucleotide encoding the polypeptide which when expressed confersresistance or susceptibility to the one or more biotrophic fungalpathogen(s), preferably a rust, a mildew or both a rust and a mildew,more preferably to one or more or all of leaf rust, stripe rust, stemrust and powdery mildew.
 21. The process of claim 20, wherein one ormore of the following apply, a) the polynucleotide comprises nucleotideshaving a sequence as provided in SEQ ID NO:2 or SEQ ID NO:3, a sequencewhich is at least 40% identical to one or both of SEQ ID NO:2 and SEQ IDNO:3, or a sequence which hybridizes to one or both of SEQ ID NO:2 andSEQ ID NO:3, b) the plant is a cereal, grape, beet or leguminous plant,c) the polypeptide is a plant polypeptide or mutant thereof, and d) stepii) further comprises stably integrating the polynucleotide operablylinked to a promoter into the genome of the plant. 22.-47. (canceled)48. A plant seed comprising a recombinant cell of claim
 1. 49.(canceled)
 50. A method of producing a plant part, the methodcomprising, a) growing a plant of claim 12, and b) harvesting the plantpart.
 51. A method of producing flour, wholemeal, starch or otherproduct obtained from seed, the method comprising; a) obtaining seed ofclaim 48, and b) extracting the flour, wholemeal, starch or otherproduct. 52.-63. (canceled)
 64. The cell of claim 1, wherein thepolypeptide comprises an amino acid sequence which is at least 90%identical to SEQ ID NO:1.
 65. A method for increasing the resistance ofa plant to one or more biotrophic fungal pathogen(s), the methodcomprising crossing a first plant to a second plant, wherein the firstplant is the plant of claim
 12. 66. A plant comprising a Lr67 geneencoding a polypeptide, wherein the plant has increased resistance toone or more biotrophic fungal pathogen(s) compared to a wild-typevariety of the plant, and wherein the Lr67 gene is a mutagenized orrecombinant polynucleotide.